Carboxyesterase polypeptides for amide coupling

ABSTRACT

The present invention provides engineered carboxyesterase enzymes having improved properties as compared to a naturally occurring wild-type carboxyesterase enzymes, as well as polynucleotides encoding the engineered carboxyesterase enzymes, host cells capable of expressing the engineered carboxyesterase enzymes, and methods of using the engineered carboxyesterase enzymes in amidation reactions.

The present application is a divisional of co-pending U.S. patentapplication Ser. No. 16/218,003, filed Dec. 12, 2018, which claimspriority to U.S. Prov. Pat. Appln. Ser. No. 62/598,189, filed Dec. 13,2017, which is hereby incorporated by reference in its entirety for allpurposes.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing concurrently submitted herewith under 37 C.F.R. §1.821 in a computer readable form (CRF) via EFS-Web as file name,CX2-165US1_ST25.txt, is herein incorporated by reference. The electroniccopy of the Sequence Listing was created on Dec. 7, 2018, with a filesize of 420 Kbytes.

FIELD OF THE INVENTION

The present invention provides engineered carboxyesterases (E.C. 3.1.1)having improved non-native properties as compared to naturally occurringwild-type (WT) carboxyesterase enzymes, as well as polynucleotidesencoding the engineered carboxyesterase enzymes, host cells capable ofexpressing the engineered carboxyesterase enzymes, and methods ofapplying the engineered carboxyesterase enzymes to amidation reactions.

BACKGROUND OF THE INVENTION

Amide bonds are key functional moieties in various synthetic molecules,including polymers (e.g., proteins, nylon), pesticides (e.g., propanil,chlorpropham), and pharmaceuticals (e.g., valsartan, lisdexamfetamine).A recent survey of the prevalence of reaction types employed in thepursuit of novel drug candidates listed N-acyl amidation atapproximately 16% among all of those reactions. (Roughley and Jordan, J.Med. Chem., 54: 3451-3479 [2011]). When produced using traditionalchemical methods, amide bond formation is a resource intensivetransformation. Amide bonds are typically synthesized from carboxylicacids and amines. However, the reaction between these two functionalgroups does not occur spontaneously at ambient temperatures, with thenecessary elimination of water only occurring at high temperatures(e.g., 200° C.). These conditions tend to be detrimental to thesubstrates and products.

For amidation to occur under more suitable conditions, activation of acarboxylic acid is generally required in order to couple to an amine.Carboxylic activation usually occurs with the aid of a coupling reagentto form an activated ester or anhydride or by transforming thecarboxylic acid into the corresponding acid chloride (i.e., through theSchotten-Baumann reaction; See El-Faham and Albericio, Chem. Rev., 111:6557-6602 [2011]). These reactions are performed with super- orstoichiometric concentrations of expensive coupling reagents thatutilize atom-inefficient synthetic routes (See, Pattabiraman and Bode,Nature, 480: 471-479 [2011]). In addition, the reagents, as well as theresulting waste, can be highly toxic and environmentally unfriendly. Atleast one equivalent of waste is produced per product molecule formed inthese reactions, resulting in very low atom economy. Removal of thewaste from the reaction mixture is a tedious and expensive process.Thus, more efficient and environmentally friendly means are needed inthe art for the production of amide bonds in various settings.

Lipases have found application on commercial scale for hydrolysis offatty acid esters, and have been employed for amidation of esters (SeemFaber, Biotransformations in Organic Chemistry, In Special Techniques,Springer-Verlag, New York, N.Y., [2011]; and Kalkote, et al., Asian J.Biochem., 2: 279-283 [2007]). The formation of amide bonds using enzymesis a highly atom economical process, as there is no need to activate thecarboxylic acid as under typical coupling approaches. The use of enzymesin these reactions provides great industrial value, as they areenvironmentally friendly, occur under mild conditions, and are lessexpensive than the currently available chemical routes. While eukaryoticproteases and lipases are capable of forming amide bonds (See, Adolfssonet al., Chem. Soc. Rev., 43: 2714-2742 [2014]; Guzman, et al., Elec. J.Biotech., 10(2) [2007]; and Asano et al., J. Biosci. Bioeng.,100(6):662-666 [2005]), these enzymes are typically poorly expressed inprokaryotic expression systems and may perform inferiorly in organicsolvents. In contrast, with the aid of directed evolution, thecarboxyesterases of the present invention have been easily produced inprokaryotic (E. coli) expression systems and have increased toleranceand practicality in organic solvent regimes more suited to this reactiontype.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a sequence alignment of the polynucleotide sequenceencoding the E. coli codon optimized for wild-type carboxyesteraseenzyme, Thermobifida fusca (T. fusca) (SEQ ID NO: 1) against each of thepolynucleotide sequences that encode the engineered carboxyesterasesequences shown in the Sequence Listing filed herewith (SEQ ID NOs: 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135).All of these disclosed polynucleotide sequences are between 98.9-99.9%identical to each other.

FIG. 2 shows a sequence alignment of the polypeptide sequence derivedfrom the wild-type carboxyesterase enzyme, Thermobifida fusca (T. fusca)(SEQ ID NO: 2) against each of the engineered polypeptidecarboxyesterase sequences shown in the Sequence Listing filed herewith(SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, and 136). All of these disclosed polypeptide sequences arebetween 98.6-99.8% identical to each other.

SUMMARY OF THE INVENTION

The present invention provides engineered carboxyesterases (E.C. 3.1.1)having improved non-native properties as compared to naturally occurringwild-type (WT) carboxyesterase enzymes, as well as polynucleotidesencoding the engineered carboxyesterase enzymes, host cells capable ofexpressing the engineered carboxyesterase enzymes, and methods ofapplying the engineered carboxyesterase enzymes to amidation reactions.

The present invention provides engineered carboxyesterases comprisingpolypeptide sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identityto SEQ ID NO: 2 or a functional fragment thereof, wherein the engineeredcarboxyesterases comprise at least one substitution or substitution setin their polypeptide sequences, and wherein the amino acid positions ofthe polypeptide sequences are numbered with reference to SEQ ID NO: 2.In some embodiments, at least one substitution or substitution set inthe polypeptide sequence comprises substitutions at positions selectedfrom 39, 39/323, 62, 62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263, 77,77/184, 103, 103/147, 104, 104/429, 105, 107, 107/185, 108, 109,109/117, 110, 111, 113, 114, 115, 117, 118, 118/269, 118/349, 119, 126,147, 153, 153/215, 164, 164/271, 174, 174/282, 183, 184, 184/249, 185,186, 187, 188, 190, 209, 210, 211, 212, 213, 213/271, 213/345, 214, 215,215/271, 216, 217, 217/231, 224, 224/268/372, 231, 249, 249/284, 263,268, 269, 270, 270/470, 271, 271/416, 276, 277, 278, 279, 279/280/282,280, 281, 281/374, 282, 283, 283/429, 284, 284/438, 285, 286, 311, 317,320, 320/323, 320/323/372, 320/372/376, 320/376/377, 321, 323, 324, 345,349, 372, 372/376, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429,438, and 470, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 2. In some additionalembodiments, at least one substitution or substitution set in thepolypeptide sequence comprises substitutions selected from: 39/323,62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263, 77/184, 103, 103/147,104, 104/429, 105, 107, 107/185, 108, 109/117, 110, 111, 113, 114, 115,117, 118, 118/269, 118/349, 119, 126, 153, 153/215, 164/271, 174/282,183, 184, 184/249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213,213/271, 213/345, 214, 215, 215/271, 216, 217, 217/231, 224/268/372,249/284, 269, 270, 270/470, 271, 271/416, 276, 277, 278, 279,279/280/282, 280, 281, 281/374, 282, 283, 283/429, 284, 284/438, 285,286, 311, 317, 320, 320/323, 320/323/372, 320/372/376, 320/376/377, 321,323, 324, 372, 372/376, 373, 376, 377, 405, 420, 427, 428, and 429,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some additional embodiments,at least one substitution or substitution set in the polypeptidesequence comprises substitutions selected from: 39M/323I, 62H/117G, 63A,63R, 63T, 63Y, 64A, 64E, 64G, 64I, 64T, 64V, 64W, 65G, 65S, 65T, 65W,66N, 68L, 68P, 69F, 69G, 69H, 69L, 69V, 69W, 69Y, 70L, 70R, 70T, 70W,71F, 71G, 71H/263R, 71P, 71R, 71V, 71Y, 77S/184G, 103P, 103R, 103T/147S,104P, 104Q/429V, 105L, 107D/185W, 107L, 107P, 107S, 108G, 108K, 108Q,108R, 108S, 108W, 109G/117M, 110A, 110H, 110P, 110S, 111L, 111M, 111R,111S, 111V, 111W, 113P, 114A, 114H, 114Q, 115H, 115T, 115V, 117A, 117F,118G/349V, 118I, 118N, 118N/269T, 119G, 119P, 119S, 126C, 153H/215P,153L, 164R/271T, 174D/282V, 183P, 184F, 184G, 184P, 184S/249T, 184Y,185A, 185T, 186C, 186G, 186P, 186R, 186T, 187P, 188E, 188G, 190H, 190K,190L, 190M, 190Q, 190R, 190W, 209E, 209G, 209P, 209S, 209V, 210P, 210T,210W, 211I, 211L, 211R, 211V, 212A, 212P, 212R, 212S, 213C, 213E, 213L,213N, 213P, 213Q, 213R/345G, 213S, 213T/271K, 213V, 214K, 214L, 214T,214V, 215K, 215M, 215P, 215R, 215R/271R, 215W, 216P, 217G, 217L, 217P,217R, 217R/231V, 217S, 217V, 217W, 224I/268S/372F, 249V/284P, 269N,269V, 270I, 270I/470M, 270R, 271A, 271K, 271L, 271P, 271Q/416V, 271S,271T, 276F, 277M, 278H, 278S, 279C, 279E, 279G, 279L/280G/282M, 279V,280E, 280G, 280S, 281P, 281V, 281Y/374N, 282A, 282C, 282Q, 282R, 282S,282T, 282W, 283C, 283D, 283K, 283R/429V, 283T, 283V, 283Y, 284C, 284T,284T/438T, 284V, 285L, 285M, 285P, 286V, 311I, 317C, 317P, 320A, 320F,320G, 320G/323S, 320S, 320S/323S/372A, 320S/372A/376G, 320S/376G/377V,320W, 321L, 321S, 323C, 323I, 323R, 323Y, 324A, 372A/376A, 372L, 373G,376A, 376G, 376L, 376M, 377L, 377W, 377Y, 405D, 420G, 427A, 428V, and429L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some further embodiments, atleast one substitution or substitution set in the polypeptide sequencecomprises substitutions selected from: T39M/F323I, R62H/P117G, P63A,P63R, P63T, P63Y, P64A, P64E, P64G, P64I, P64T, P64V, P64W, Y65G, Y65S,Y65T, Y65W, P66N, A68L, A68P, I69F, I69G, I69H, I69L, I69V, I69W, I69Y,G70L, G70R, G70T, G70W, A71F, A71G, A71H/Q263R, A71P, A71R, A71V, A71Y,F77S/E184G, W103P, W103R, W103T/P147S, I104P, I104Q/A429V, H105L,G107D/S185W, G107L, G107P, G107S, A108G, A108K, A108Q, A108R, A108S,A108W, F109G/P117M, T110A, T110H, T110P, T110S, N111L, N111M, N111R,N111S, N111V, N111W, S113P, G114A, G114H, G114Q, S115H, S115T, S115V,P117A, P117F, V118G/A349V, V118I, V118N, V118N/A269T, Y119G, Y119P,Y119S, R126C, R153H/N215P, R153L, W164R/W271T, G174D/L282V, G183P,E184F, E184G, E184P, E184S/A249T, E184Y, S185A, S185T, A186C, A186G,A186P, A186R, A186T, G187P, A188E, A188G, S190H, S190K, S190L, S190M,S190Q, S190R, S190W, L209E, L209G, L209P, L209S, L209V, Q210P, Q210T,Q210W, S211I, S211L, S211R, S211V, G212A, G212P, G212R, G212S, A213C,A213E, A213L, A213N, A213P, A213Q, A213R/S345G, A213S, A213T/W271K,A213V, G214K, G214L, G214T, G214V, N215K, N215M, N215P, N215R,N215R/W271R, N215W, M216P, A217G, A217L, A217P, A217R, A217R/A231V,A217S, A217V, A217W, T224I/P268S/I372F, A249V/F284P, A269N, A269V,V270I, V270I/V470M, V270R, W271A, W271K, W271L, W271P, W271Q/A416V,W271S, W271T, A276F, G277M, G278H, G278S, S279C, S279E, S279G,S279L/V280G/L282M, S279V, V280E, V280G, V280S, L281P, L281V,L281Y/D374N, L282A, L282C, L282Q, L282R, L282S, L282T, L282W, P283C,P283D, P283K, P283R/A429V, P283T, P283V, P283Y, F284C, F284T,F284T/P438T, F284V, A285L, A285M, A285P, P286V, L311I, T317C, T317P,Y320A, Y320F, Y320G, Y320G/F323S, Y320S, Y320S/F323S/I372A,Y320S/I372A/V376G, Y320S/V376G/F377V, Y320W, R321L, R321S, F323C, F323I,F323R, F323Y, L324A, I372A/V376A, I372L, T373G, V376A, V376G, V376L,V376M, F377L, F377W, F377Y, P405D, P420G, D427A, R428V, and A429L,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some embodiments, theengineered carboxyesterases comprise a substitution at position 282,wherein the position is numbered with reference to SEQ ID NO: 2. In somefurther embodiments, the substitution at position 282 is aliphatic,non-polar, basic, polar, or aromatic. In yet some additionalembodiments, the substitution is selected from: X282T, X282G, X282A,X282V, X282M, X282C, X282W, X282Q, X282S, X282T, and X282R.

The present invention also provides engineered carboxyesterasescomprising a polypeptide sequences having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 8 or a functional fragment thereof,wherein the engineered carboxyesterases comprises at least onesubstitution or substitution set in the polypeptide sequences, whereinthe amino acid positions of the polypeptide sequences are numbered withreference to SEQ ID NO: 8. In some embodiments, at least onesubstitution or substitution set in the polypeptide sequence comprisessubstitutions at positions selected from: 63, 63/65/108, 63/65/108/189,63/65/108/377, 63/65/282/285/320/323, 63/65/320/323, 63/108,63/108/282/285/377, 63/108/285/377, 63/108/320/323, 63/189, 63/212/215,63/212/215/268/269/343, 63/215, 63/215/269, 63/215/270/271, 63/215/343,63/268/269/270/429, 63/377, 65/69/70/281/372, 65/69/70/372, 65/69/372,65/70/372, 65/320, 65/320/323, 68, 68/69/189/214, 68/69/189/214/215,68/69/189/214/215/271, 68/69/189/214/215/271/281/282/343/381,68/69/189/214/271/280, 68/69/189/214/372, 68/69/189/214/377/381,68/69/189/271, 68/69/189/271/280/372/381, 68/69/189/280/281/282/372/377,68/69/189/281/282/372/377/381, 68/69/189/343/381, 68/69/189/372,68/69/189/381, 68/69/214/215/271, 68/69/214/343, 68/69/215, 68/69/271,68/69/282/287, 68/69/343/372, 68/108/377, 68/184, 68/184/189,68/189/271/372, 68/189/343, 68/214/215/271/281/282/372,68/215/271/343/372/381, 68/215/377, 68/271/372, 68/377, 69, 69/70,69/70/331/372, 69/70/372, 69/70/459, 69/108, 69/108/270/372/377,69/108/281/285, 69/110/215/281, 69/189, 69/189/214/271/281/282/343,69/189/214/343/372, 69/189/215/343, 69/189/271, 69/189/271/281/282,69/189/271/343, 69/189/271/343/381, 69/189/280/282/343/372/381,69/189/281/373, 69/189/282, 69/189/343/381, 69/189/372, 69/189/372/377,69/189/377, 69/212/213/215/280/281, 69/214/215/271/372/377/381,69/214/271/282, 69/214/271/343, 69/215, 69/215/269/270/377,69/215/271/280/281/282, 69/215/271/282, 69/215/271/372, 69/215/285/317,69/215/323, 69/215/343, 69/215/343/372/381, 69/271, 69/271/372, 69/282,69/282/343/372, 69/285/373, 69/372, 70, 70/212, 108, 108/189,108/189/282/285/320, 108/189/320, 108/189/377, 108/215, 108/215/377,108/269/270, 108/270, 108/282/285/377, 108/285, 108/320/323, 108/377,126, 126/184/213/280/281/285/320, 126/184/213/372, 126/189/285/372,126/215, 126/372, 181/215, 189, 189/214, 189/214/215/271/282, 189/215,189/215/249/277, 189/215/271/281/282/377, 189/215/343/372, 189/270/285,189/270/372, 189/280/282, 189/320/377, 189/343, 189/343/377,189/372/377, 189/377, 189/381, 213/215/320, 214/215/271,214/215/271/377, 214/271, 214/280/282/343/377/381, 215,215/249/280/281/285/372, 215/271/372, 215/280/281/285/372,215/281/285/372, 215/281/285/373, 215/281/373, 215/285, 215/285/317,215/285/346, 215/285/445, 215/320, 215/320/372, 215/323, 215/372,215/372/377, 215/373, 215/377, 215/381, 249/377, 269/270/281/372/377,270/377, 271, 271/343, 271/343/372, 271/343/372/381, 280/285/372,281/372, 282/285/320/323, 285/323, 320, 343/372, 372, 372/377, 372/381,373, and 377, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 8. In some furtherembodiments, at least one substitution or substitution set in thepolypeptide sequence comprises at least one substitution selected from:63A, 63A/189A, 63A/215R/343V, 63R, 63R/65G/108G, 63R/65G/108G/189L,63R/65G/108G/377I, 63R/65G/282A/285L/320W/323I, 63R/65G/320W/323I,63R/108G, 63R/108G/282A/285L/377L, 63R/108G/285L/377I,63R/108G/320W/323C, 63R/377I, 63T/215R, 63Y, 63Y/189L, 63Y/212P/215R,63Y/212P/215R/268A/269N/343V, 63Y/215P/269N, 63Y/215R,63Y/215R/270I/271S, 63Y/268A/269N/270I/429V, 65G/320W, 65G/320W/323I,65W/69L/372L, 65W/69M/70A/281P/372L, 65W/69W/70L/372L, 65W/70L/372M,68P, 68P/69L/189E/214R/271Y/280G, 68P/69L/189E/214R/372L,68P/69L/189I/214R/215P/271Y, 68P/69L/189I/281P/282C/372L/377Y/381L,68P/69L/189Q/214R, 68P/69L/189Q/271Y/280G/372L/381L, 68P/69L/215P,68P/69L/271Y, 68P/69L/282C/287I, 68P/69L/343V/372L,68P/69W/189E/214R/215P/271Y/281P/282G/343V/381L,68P/69W/189E/280G/281P/282A/372L/377Y, 68P/69W/189E/343V/381L,68P/69W/189I/214R/215P, 68P/69W/189I/214R/377Y/381L, 68P/69W/189I/271Y,68P/69W/189I/372L, 68P/69W/189I/381L, 68P/69W/214R/215P/271Y,68P/69W/214R/343V, 68P/69W/215P, 68P/108G/377L, 68P/184S, 68P/184S/189E,68P/189I/271Y/372L, 68P/189I/343V, 68P/214R/215P/271Y/281P/282A/372L,68P/215P/271Y/343V/372L/381L, 68P/215P/377L, 68P/271Y/372L, 68P/377L,69F/108G/270E/372L/377L, 69F/189L, 69F/215K, 69F/215K/269L/270I/377L,69F/215R, 69F/285L/373G, 69L, 69L/70L/331Q/372M,69L/189E/271Y/281P/282A, 69L/189I, 69L/189I/214R/271Y/281P/282A/343V,69L/189I/271Y/343V/381L, 69L/189I/280G/282G/343V/372L/381L,69L/189I/282A, 69L/189Q/377Y, 69L/215P/271Y/280G/281P/282C,69L/215P/271Y/282A, 69L/215P/271Y/372L, 69L/215P/343V/372L/381L,69L/215R/285P/317P, 69L/271Y, 69L/271Y/372L, 69L/282C/343V/372L,69L/372L, 69M/70A/372M, 69W, 69W/70L, 69W/70L/372M, 69W/70L/459R,69W/108S, 69W/189E/214R/343V/372L, 69W/189E/271Y/343V, 69W/189E/372L,69W/189I, 69W/189I/215P/343V, 69W/189I/271Y, 69W/189I/343V/381L,69W/189Q/372L/377Y, 69W/212A/213L/215R/280G/281P,69W/214R/215P/271Y/372L/377Y/381L, 69W/214R/271Y/282A,69W/214R/271Y/343V, 69W/215K/343V, 69W/215P, 69W/215R, 69W/215R/323Y,69W/282A, 69W/372M, 69Y/108G/281P/285P, 69Y/110A/215R/281P,69Y/189L/281P/373G, 70L, 70L/212P, 108G, 108G/189I/282A/285L/320W,108G/189L, 108G/189L/320W, 108G/189L/377I, 108G/215K, 108G/215P/377L,108G/269L/270E, 108G/270E, 108G/282A/285L/377L, 108G/285L,108G/320W/323I, 108G/377I, 108G/377L, 126C,126C/184S/213S/280G/281P/285L/320G, 126C/184S/213S/372L,126C/189I/285L/372L, 126C/215P, 126C/372L, 181L/215P, 189E/372L/377Y,189I, 189I/214R/215P/271Y/282G, 189I/215K, 189I/215P/343V/372L,189I/215R/249T/277M, 189I/270E/285L, 189I/270E/372L, 189I/280G/282A,189I/320W/377I, 189I/343V, 189I/377I, 189L, 189Q, 189Q/214R,189Q/215P/271Y/281P/282C/377Y, 189Q/343V, 189Q/343V/377Y, 189Q/381L,213S/215P/320G, 214R/215P/271Y, 214R/215P/271Y/377Y, 214R/271Y,214R/280G/282A/343V/377Y/381L, 215K, 215K/281P/285L/372L,215K/281P/373G, 215K/285L/317P, 215K/285L/445L, 215K/323Y, 215K/372L,215K/372L/377L, 215K/373G, 215P, 215P/271Y/372L, 215P/320G,215P/320G/372L, 215P/372L, 215P/372L/377L, 215P/377L, 215P/381L, 215R,215R/249T/280G/281P/285L/372L, 215R/280G/281P/285L/372L,215R/281P/285L/373G, 215R/285P, 215R/320G, 215R/372L, 215W,215W/285L/346S, 215W/285P, 215W/373G, 249T/377L,269L/270E/281P/372L/377L, 270E/377L, 271Y, 271Y/343V, 271Y/343V/372L,271Y/343V/372L/381L, 280G/285L/372L, 281P/372L, 282A/285L/320W/323I,285L/323I, 320W, 343V/372L, 372L, 372L/377L, 372L/381L, 372M, 373G, and377L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 8. In some further embodiments, atleast one substitution or substitution set in the polypeptide sequencecomprises at least one substitution selected from: P63A, P63A/M189A,P63A/N215R/A343V, P63R, P63R/Y65G/A108G, P63R/Y65G/A108G/M189L,P63R/Y65G/A108G/F377I, P63R/Y65G/T282A/A285L/Y320W/F323I,P63R/Y65G/Y320W/F323I, P63R/A108G, P63R/A108G/T282A/A285L/F377L,P63R/A108G/A285L/F377I, P63R/A108G/Y320W/F323C, P63R/F377I, P63T/N215R,P63Y, P63Y/M189L, P63Y/G212P/N215R, P63Y/G212P/N215R/P268A/A269N/A343V,P63Y/N215P/A269N, P63Y/N215R, P63Y/N215R/V270I/W271S,P63Y/P268A/A269N/V270I/A429V, Y65G/Y320W, Y65G/Y320W/F323I,Y65W/I69L/I372L, Y65W/I69M/G70A/L281P/I372L, Y65W/I69W/G70L/I372L,Y65W/G70L/I372M, A68P, A68P/I69L/M189E/G214R/W271Y/V280G,A68P/I69L/M189E/G214R/I372L, A68P/I69L/M189I/G214R/N215P/W271Y,A68P/I69L/M189I/L281P/T282C/I372L/F377Y/A381L, A68P/I69L/M189Q/G214R,A68P/I69L/M189Q/W271Y/V280G/I372L/A381L, A68P/I69L/N215P,A68P/I69L/W271Y, A68P/I69L/T282C/V287I, A68P/I69L/A343V/I372L,A68P/I69W/M189E/G214R/N215P/W271Y/L281P/T282G/A343V/A381L,A68P/I69W/M189E/V280G/L281P/T282A/I372L/F377Y,A68P/I69W/M189E/A343V/A381L, A68P/I69W/M189I/G214R/N215P,A68P/I69W/M189I/G214R/F377Y/A381L, A68P/I69W/M189I/W271Y,A68P/I69W/M189I/I372L, A68P/I69W/M189I/A381L,A68P/I69W/G214R/N215P/W271Y, A68P/I69W/G214R/A343V, A68P/I69W/N215P,A68P/A108G/F377L, A68P/E184S, A68P/E184S/M189E, A68P/M189I/W271Y/I372L,A68P/M189I/A343V, A68P/G214R/N215P/W271Y/L281P/T282A/I372L,A68P/N215P/W271Y/A343V/I372L/A381L, A68P/N215P/F377L, A68P/W271Y/I372L,A68P/F377L, I69F/A108G/V270E/I372L/F377L, I69F/M189L, I69F/N215K,I69F/N215K/A269L/V270I/F377L, I69F/N215R, I69F/A285L/T373G, I69L,I69L/G70L/P331Q/I372M, I69L/M189E/W271Y/L281P/T282A, I69L/M189I,I69L/M189I/G214R/W271Y/L281P/T282A/A343V, I69L/M189I/W271Y/A343V/A381L,I69L/M189I/V280G/T282G/A343V/I372L/A381L, I69L/M189I/T282A,I69L/M189Q/F377Y, I69L/N215P/W271Y/V280G/L281P/T282C,I69L/N215P/W271Y/T282A, I69L/N215P/W271Y/I372L,I69L/N215P/A343V/I372L/A381L, I69L/N215R/A285P/T317P, I69L/W271Y,I69L/W271Y/I372L, I69L/T282C/A343V/I372L, I69L/I372L, I69M/G70A/I372M,I69W, I69W/G70L, I69W/G70L/I372M, I69W/G70L/G459R, I69W/A108S,I69W/M189E/G214R/A343V/I372L, I69W/M189E/W271Y/A343V, I69W/M189E/I372L,I69W/M189I, I69W/M189I/N215P/A343V, I69W/M189I/W271Y,I69W/M189I/A343V/A381L, I69W/M189Q/I372L/F377Y,I69W/G212A/A213L/N215R/V280G/L281P,I69W/G214R/N215P/W271Y/I372L/F377Y/A381L, I69W/G214R/W271Y/T282A,I69W/G214R/W271Y/A343V, I69W/N215K/A343V, I69W/N215P, I69W/N215R,I69W/N215R/F323Y, I69W/T282A, I69W/I372M, I69Y/A108G/L281P/A285P,I69Y/T110A/N215R/L281P, I69Y/M189L/L281P/T373G, G70L, G70L/G212P, A108G,A108G/M189I/T282A/A285L/Y320W, A108G/M189L, A108G/M189L/Y320W,A108G/M189L/F377I, A108G/N215K, A108G/N215P/F377L, A108G/A269L/V270E,A108G/V270E, A108G/T282A/A285L/F377L, A108G/A285L, A108G/Y320W/F323I,A108G/F377I, A108G/F377L, R126C,R126C/E184S/A213S/V280G/L281P/A285L/Y320G, R126C/E184S/A213S/I372L,R126C/M189I/A285L/I372L, R126C/N215P, R126C/I372L, V181L/N215P,M189E/I372L/F377Y, M189I, M189I/G214R/N215P/W271Y/T282G, M189I/N215K,M189I/N215P/A343V/I372L, M189I/N215R/A249T/G277M, M189I/V270E/A285L,M189I/V270E/I372L, M189I/V280G/T282A, M189I/Y320W/F377I, M189I/A343V,M189I/F377I, M189L, M189Q, M189Q/G214R,M189Q/N215P/W271Y/L281P/T282C/F377Y, M189Q/A343V, M189Q/A343V/F377Y,M189Q/A381L, A213S/N215P/Y320G, G214R/N215P/W271Y,G214R/N215P/W271Y/F377Y, G214R/W271Y,G214R/V280G/T282A/A343V/F377Y/A381L, N215K, N215K/L281P/A285L/I372L,N215K/L281P/T373G, N215K/A285L/T317P, N215K/A285L/V445L, N215K/F323Y,N215K/I372L, N215K/I372L/F377L, N215K/T373G, N215P, N215P/W271Y/I372L,N215P/Y320G, N215P/Y320G/I372L, N215P/I372L, N215P/I372L/F377L,N215P/F377L, N215P/A381L, N215R, N215R/A249T/V280G/L281P/A285L/I372L,N215R/V280G/L281P/A285L/I372L, N215R/L281P/A285L/T373G, N215R/A285P,N215R/Y320G, N215R/I372L, N215W, N215W/A285L/G346S, N215W/A285P,N215W/T373G, A249T/F377L, A269L/V270E/L281P/I372L/F377L, V270E/F377L,W271Y, W271Y/A343V, W271Y/A343V/I372L, W271Y/A343V/I372L/A381L,V280G/A285L/I372L, L281P/I372L, T282A/A285L/Y320W/F323I, A285L/F323I,Y320W, A343V/I372L, I372L, I372L/F377L, I372L/A381L, I372M, T373G, andF377L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 8.

The present invention also provides engineered carboxyesterasescomprising polypeptide sequences having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 138 or a functional fragment thereof,wherein the engineered carboxyesterases comprise at least onesubstitution or substitution set in their polypeptide sequences, andwherein the amino acid positions of the polypeptide sequences arenumbered with reference to SEQ ID NO: 138.

The present invention also provides engineered carboxyesterasescomprising polypeptide sequences having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequenceidentity to SEQ ID NO: 140 or a functional fragment thereof, wherein theengineered carboxyesterases comprise at least one substitution orsubstitution set in the polypeptide sequences, and wherein the aminoacid positions of the polypeptide sequences are numbered with referenceto SEQ ID NO: 140.

The present invention also provides engineered carboxyesterasescomprising polypeptide sequences selected from: SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136.

The present invention further provides engineered carboxyesterasesexhibiting at least one improved property as compared to the wild-typeT. fusca carboxyesterase of SEQ ID NO:2. In some embodiments, theimproved property is selected from: improved amidation activity, solventtolerance, thermostability, pH stability, regiosteroselectivity,stereoselectivity, substrate scope, and/or reduced substrate or productinhibition, and reduced toxicity to bacterial host cells producing theengineered carboxyesterase. In some additional embodiments, thebacterial host cells comprise E. coli. In still some additionalembodiments, the engineered carboxyesterases exhibit improved solventtolerance to at least one solvent selected from: acetone, acetonitrile,toluene, tetrahydrofuran, isopropanol, isopropyl acetate, dimethylsulfoxide and/or methyl ethyl ketone. In some further embodiments, theengineered carboxyesterases exhibit greater activity than wild-type T.fusca carboxyesterase on at least one substrate selected from: aniline,isobutylamine, n-butylamine, t-butylamine,N′-t-butoxycarbonyl-benzhydrazide, 4-methylpiperidine,O-t-butylhdroxylamine, benzylamine, 2,6-dimethylaniline,(S)-(−)-α-methylbenzylamine, (R)-(+)-α-methylbenzylamine, methylphenylacetate, ethyl acetate, ethyl benzoate, 2-pyrazinyl ethyl ester,4-ethyl-1H-indole ester, N,N-diethylglycyl methyl ester. In someadditional embodiments, the engineered carboxyesterases exhibit greateractivity than wild-type T. fusca carboxyesterase on at least onesubstrate or substrate set selected from aniline, isobutylamine,n-butylamine, t-butylamine, N′-t-butoxycarbonyl-benzhydrazide,4-methylpiperidine, O-t-butylhdroxylamine, benzylamine,2,6-dimethylaniline, (S)-(−)-α-methylbenzylamine,(R)-(+)-α-methylbenzylamine, methyl phenylacetate, ethyl acetate, ethylbenzoate, 2-pyrazinyl ethyl ester, 4-ethyl-1H-indole ester,N,N-diethylglycyl methyl ester. In yet some further embodiments, theengineered carboxyesterases exhibit greater activity than wild-type T.fusca carboxyesterase in producing at least one product selected from:acetanilide, N-n-butyl-benzylacetamide,N-[(S)-1-phenylethyl]-pyrazinylamide, N-[(S)-1-phenylethyl]-benzamide,N-[(R)-1-phenylethyl]-benzamide, N′-t-butoxycarbonyl-benzhydrazide,1-benzoyl-4-methylpiperidine, 2-pyrazinyl-4-methylpiperidine,N-isobutyl-benzamide, N-t-butyl-benzamide, N-t-butylhydroxyl-benzamide,N-isobutyl-1H-indol-4-amide, N′,N′-(diethylamino)-N-phenylacetamide,N′,N′-(diethylamino)-N-benzylacetamide,N′,N′-(diethylamino)-N-2,6-dimethylphenylacetamide (i.e., lidocaine). Insome embodiments, the engineered carboxyesterases of the inventioncomprises at least one substitution selected from: X343V, X372L,X320W/G, X214R, X282C, X271Y, X65G, wherein the substitutions arenumbered with reference to SEQ ID NO:2, and wherein the engineeredcarboxyesterase exhibits greater activity than wild-type T. fuscacarboxyesterase on a hindered amine for formation ofN′,N′-(diethylamino)-N-2,6-dimethylphenylacetamide from ethyl benzoateand 2,6-dimethylaniline, as shown in the following schematic.

In yet some additional embodiments, the engineered carboxyesterasescomprise at least one substitution selected from: X268A, X63A/R,X189Q/I/E, X214R, X282G/C, X381L, and X69W, wherein the substitutionsare numbered with reference to SEQ ID NO:2, and wherein the engineeredcarboxyesterase exhibits greater activity than wild-type T. fuscacarboxyesterase on a secondary amine for formation of1-benzoyl-4-methyl-piperidine from ethyl benzoate and4-methyl-piperidine, as shown in the following schematic.

In yet some additional embodiments, the engineered carboxyesterasesprovided herein are purified. In still some further embodiments, theengineered carboxyesterases are immobilized. The present invention alsoprovides compositions comprising at least one engineered carboxyesteraseprovided herein.

The present invention also provides polynucleotide sequences encoding atleast one engineered carboxyesterase provided herein. In someembodiments, the polynucleotide sequences encode at least one engineeredcarboxyesterase comprising a polypeptide sequence having at least 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 2 or a functional fragmentthereof, wherein the engineered carboxyesterase comprises at least onesubstitution or substitution set in its polypeptide sequence, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2. In some embodiments, the polynucleotidesequences encode at least one engineered carboxyesterase comprisingsubstitutions at positions selected from: 39, 39/323, 62, 62/117, 63,64, 65, 66, 68, 69, 70, 71, 71/263, 77, 77/184, 103, 103/147, 104,104/429, 105, 107, 107/185, 108, 109, 109/117, 110, 111, 113, 114, 115,117, 118, 118/269, 118/349, 119, 126, 147, 153, 153/215, 164, 164/271,174, 174/282, 183, 184, 184/249, 185, 186, 187, 188, 190, 209, 210, 211,212, 213, 213/271, 213/345, 214, 215, 215/271, 216, 217, 217/231, 224,224/268/372, 231, 249, 249/284, 263, 268, 269, 270, 270/470, 271,271/416, 276, 277, 278, 279, 279/280/282, 280, 281, 281/374, 282, 283,283/429, 284, 284/438, 285, 286, 311, 317, 320, 320/323, 320/323/372,320/372/376, 320/376/377, 321, 323, 324, 345, 349, 372, 372/376, 373,374, 376, 377, 405, 416, 420, 427, 428, 429, 438, and 470, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2. In some additional embodiments, thepolynucleotide sequences encode at least one engineered carboxyesterasecomprising at least one substitution or substitution set selected from:39/323, 62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263, 77/184, 103,103/147, 104, 104/429, 105, 107, 107/185, 108, 109/117, 110, 111, 113,114, 115, 117, 118, 118/269, 118/349, 119, 126, 153, 153/215, 164/271,174/282, 183, 184, 184/249, 185, 186, 187, 188, 190, 209, 210, 211, 212,213, 213/271, 213/345, 214, 215, 215/271, 216, 217, 217/231,224/268/372, 249/284, 269, 270, 270/470, 271, 271/416, 276, 277, 278,279, 279/280/282, 280, 281, 281/374, 282, 283, 283/429, 284, 284/438,285, 286, 311, 317, 320, 320/323, 320/323/372, 320/372/376, 320/376/377,321, 323, 324, 372, 372/376, 373, 376, 377, 405, 420, 427, 428, and 429,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some additional embodiments,the polynucleotide sequences encode at least one engineeredcarboxyesterase comprising at least one substitution or substitution setselected from: 39M/323I, 62H/117G, 63A, 63R, 63T, 63Y, 64A, 64E, 64G,64I, 64T, 64V, 64W, 65G, 65S, 65T, 65W, 66N, 68L, 68P, 69F, 69G, 69H,69L, 69V, 69W, 69Y, 70L, 70R, 70T, 70W, 71F, 71G, 71H/263R, 71P, 71R,71V, 71Y, 77S/184G, 103P, 103R, 103T/147S, 104P, 104Q/429V, 105L,107D/185W, 107L, 107P, 107S, 108G, 108K, 108Q, 108R, 108S, 108W,109G/117M, 110A, 110H, 110P, 110S, 111L, 111M, 111R, 111S, 111V, 111W,113P, 114A, 114H, 114Q, 115H, 115T, 115V, 117A, 117F, 118G/349V, 118I,118N, 118N/269T, 119G, 119P, 119S, 126C, 153H/215P, 153L, 164R/271T,174D/282V, 183P, 184F, 184G, 184P, 184S/249T, 184Y, 185A, 185T, 186C,186G, 186P, 186R, 186T, 187P, 188E, 188G, 190H, 190K, 190L, 190M, 190Q,190R, 190W, 209E, 209G, 209P, 209S, 209V, 210P, 210T, 210W, 211I, 211L,211R, 211V, 212A, 212P, 212R, 212S, 213C, 213E, 213L, 213N, 213P, 213Q,213R/345G, 213S, 213T/271K, 213V, 214K, 214L, 214T, 214V, 215K, 215M,215P, 215R, 215R/271R, 215W, 216P, 217G, 217L, 217P, 217R, 217R/231V,217S, 217V, 217W, 224I/268S/372F, 249V/284P, 269N, 269V, 270I,270I/470M, 270R, 271A, 271K, 271L, 271P, 271Q/416V, 271S, 271T, 276F,277M, 278H, 278S, 279C, 279E, 279G, 279L/280G/282M, 279V, 280E, 280G,280S, 281P, 281V, 281Y/374N, 282A, 282C, 282Q, 282R, 282S, 282T, 282W,283C, 283D, 283K, 283R/429V, 283T, 283V, 283Y, 284C, 284T, 284T/438T,284V, 285L, 285M, 285P, 286V, 311I, 317C, 317P, 320A, 320F, 320G,320G/323S, 320S, 320S/323S/372A, 320S/372A/376G, 320S/376G/377V, 320W,321L, 321S, 323C, 323I, 323R, 323Y, 324A, 372A/376A, 372L, 373G, 376A,376G, 376L, 376M, 377L, 377W, 377Y, 405D, 420G, 427A, 428V, and 429L,wherein the amino acid positions are numbered with reference to SEQ IDNO: 2. In some further embodiments, the polynucleotide sequences encodeat least one engineered carboxyesterase comprising at least onesubstitution or substitution set selected from: T39M/F323I, R62H/P117G,P63A, P63R, P63T, P63Y, P64A, P64E, P64G, P64I, P64T, P64V, P64W, Y65G,Y65S, Y65T, Y65W, P66N, A68L, A68P, I69F, I69G, I69H, I69L, I69V, I69W,I69Y, G70L, G70R, G70T, G70W, A71F, A71G, A71H/Q263R, A71P, A71R, A71V,A71Y, F775/E184G, W103P, W103R, W103T/P147S, I104P, I104Q/A429V, H105L,G107D/S185W, G107L, G107P, G107S, A108G, A108K, A108Q, A108R, A108S,A108W, F109G/P117M, T110A, T110H, T110P, T110S, N111L, N111M, N111R,N111S, N111V, N111W, S113P, G114A, G114H, G114Q, S115H, S115T, S115V,P117A, P117F, V118G/A349V, V118I, V118N, V118N/A269T, Y119G, Y119P,Y119S, R126C, R153H/N215P, R153L, W164R/W271T, G174D/L282V, G183P,E184F, E184G, E184P, E184S/A249T, E184Y, S185A, S185T, A186C, A186G,A186P, A186R, A186T, G187P, A188E, A188G, S190H, S190K, S190L, S190M,S190Q, S190R, S190W, L209E, L209G, L209P, L209S, L209V, Q210P, Q210T,Q210W, S211I, S211L, S211R, S211V, G212A, G212P, G212R, G212S, A213C,A213E, A213L, A213N, A213P, A213Q, A213R/S345G, A213S, A213T/W271K,A213V, G214K, G214L, G214T, G214V, N215K, N215M, N215P, N215R,N215R/W271R, N215W, M216P, A217G, A217L, A217P, A217R, A217R/A231V,A217S, A217V, A217W, T224I/P268S/I372F, A249V/F284P, A269N, A269V,V270I, V270I/V470M, V270R, W271A, W271K, W271L, W271P, W271Q/A416V,W271S, W271T, A276F, G277M, G278H, G278S, S279C, S279E, S279G,S279L/V280G/L282M, S279V, V280E, V280G, V280S, L281P, L281V,L281Y/D374N, L282A, L282C, L282Q, L282R, L282S, L282T, L282W, P283C,P283D, P283K, P283R/A429V, P283T, P283V, P283Y, F284C, F284T,F284T/P438T, F284V, A285L, A285M, A285P, P286V, L311I, T317C, T317P,Y320A, Y320F, Y320G, Y320G/F323S, Y320S, Y320S/F323S/I372A,Y320S/I372A/V376G, Y320S/V376G/F377V, Y320W, R321L, R321S, F323C, F323I,F323R, F323Y, L324A, I372A/V376A, I372L, T373G, V376A, V376G, V376L,V376M, F377L, F377W, F377Y, P405D, P420G, D427A, R428V, and A429L,wherein the amino acids are numbered with reference to SEQ ID NO: 2. Insome embodiments, the polynucleotide sequences encode at least oneengineered carboxyesterase comprising a substitution at position 282,wherein the position is numbered with reference to SEQ ID NO: 2. In somefurther embodiments, the substitution at position 282 is aliphatic,non-polar, basic, polar, or aromatic. In yet some additionalembodiments, the substitution selected from X282T, X282G, X282A, X282V,X282M, X282C, X282W, X282Q, X282S, X282T, and X282R.

The present invention also provides polynucleotide sequences encoding atleast one engineered carboxyesterase comprising a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8 or afunctional fragment thereof, wherein the engineered carboxyesterasecomprises at least one substitution or substitution set in itspolypeptide sequence, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 8. Insome embodiments, the polynucleotide sequences encode engineeredcarboxyesterases comprising at least one substitution or substitutionset at positions selected from: 63, 63/65/108, 63/65/108/189,63/65/108/377, 63/65/282/285/320/323, 63/65/320/323, 63/108,63/108/282/285/377, 63/108/285/377, 63/108/320/323, 63/189, 63/212/215,63/212/215/268/269/343, 63/215, 63/215/269, 63/215/270/271, 63/215/343,63/268/269/270/429, 63/377, 65/69/70/281/372, 65/69/70/372, 65/69/372,65/70/372, 65/320, 65/320/323, 68, 68/69/189/214, 68/69/189/214/215,68/69/189/214/215/271, 68/69/189/214/215/271/281/282/343/381,68/69/189/214/271/280, 68/69/189/214/372, 68/69/189/214/377/381,68/69/189/271, 68/69/189/271/280/372/381, 68/69/189/280/281/282/372/377,68/69/189/281/282/372/377/381, 68/69/189/343/381, 68/69/189/372,68/69/189/381, 68/69/214/215/271, 68/69/214/343, 68/69/215, 68/69/271,68/69/282/287, 68/69/343/372, 68/108/377, 68/184, 68/184/189,68/189/271/372, 68/189/343, 68/214/215/271/281/282/372,68/215/271/343/372/381, 68/215/377, 68/271/372, 68/377, 69, 69/70,69/70/331/372, 69/70/372, 69/70/459, 69/108, 69/108/270/372/377,69/108/281/285, 69/110/215/281, 69/189, 69/189/214/271/281/282/343,69/189/214/343/372, 69/189/215/343, 69/189/271, 69/189/271/281/282,69/189/271/343, 69/189/271/343/381, 69/189/280/282/343/372/381,69/189/281/373, 69/189/282, 69/189/343/381, 69/189/372, 69/189/372/377,69/189/377, 69/212/213/215/280/281, 69/214/215/271/372/377/381,69/214/271/282, 69/214/271/343, 69/215, 69/215/269/270/377,69/215/271/280/281/282, 69/215/271/282, 69/215/271/372, 69/215/285/317,69/215/323, 69/215/343, 69/215/343/372/381, 69/271, 69/271/372, 69/282,69/282/343/372, 69/285/373, 69/372, 70, 70/212, 108, 108/189,108/189/282/285/320, 108/189/320, 108/189/377, 108/215, 108/215/377,108/269/270, 108/270, 108/282/285/377, 108/285, 108/320/323, 108/377,126, 126/184/213/280/281/285/320, 126/184/213/372, 126/189/285/372,126/215, 126/372, 181/215, 189, 189/214, 189/214/215/271/282, 189/215,189/215/249/277, 189/215/271/281/282/377, 189/215/343/372, 189/270/285,189/270/372, 189/280/282, 189/320/377, 189/343, 189/343/377,189/372/377, 189/377, 189/381, 213/215/320, 214/215/271,214/215/271/377, 214/271, 214/280/282/343/377/381, 215,215/249/280/281/285/372, 215/271/372, 215/280/281/285/372,215/281/285/372, 215/281/285/373, 215/281/373, 215/285, 215/285/317,215/285/346, 215/285/445, 215/320, 215/320/372, 215/323, 215/372,215/372/377, 215/373, 215/377, 215/381, 249/377, 269/270/281/372/377,270/377, 271, 271/343, 271/343/372, 271/343/372/381, 280/285/372,281/372, 282/285/320/323, 285/323, 320, 343/372, 372, 372/377, 372/381,373, and 377, wherein the amino acid positions are numbered withreference to SEQ ID NO: 8. In some further embodiments, thepolynucleotide sequence encodes an engineered carboxyesterase comprisingat least one substitution or substitution set selected from: 63A,63A/189A, 63A/215R/343V, 63R, 63R/65G/108G, 63R/65G/108G/189L,63R/65G/108G/377I, 63R/65G/282A/285L/320W/323I, 63R/65G/320W/323I,63R/108G, 63R/108G/282A/285L/377L, 63R/108G/285L/377I,63R/108G/320W/323C, 63R/377I, 63T/215R, 63Y, 63Y/189L, 63Y/212P/215R,63Y/212P/215R/268A/269N/343V, 63Y/215P/269N, 63Y/215R,63Y/215R/270I/271S, 63Y/268A/269N/270I/429V, 65G/320W, 65G/320W/323I,65W/69L/372L, 65W/69M/70A/281P/372L, 65W/69W/70L/372L, 65W/70L/372M,68P, 68P/69L/189E/214R/271Y/280G, 68P/69L/189E/214R/372L,68P/69L/189I/214R/215P/271Y, 68P/69L/189I/281P/282C/372L/377Y/381L,68P/69L/189Q/214R, 68P/69L/189Q/271Y/280G/372L/381L, 68P/69L/215P,68P/69L/271Y, 68P/69L/282C/287I, 68P/69L/343V/372L,68P/69W/189E/214R/215P/271Y/281P/282G/343V/381L,68P/69W/189E/280G/281P/282A/372L/377Y, 68P/69W/189E/343V/381L,68P/69W/189I/214R/215P, 68P/69W/189I/214R/377Y/381L, 68P/69W/189I/271Y,68P/69W/189I/372L, 68P/69W/189I/381L, 68P/69W/214R/215P/271Y,68P/69W/214R/343V, 68P/69W/215P, 68P/108G/377L, 68P/184S, 68P/184S/189E,68P/189I/271Y/372L, 68P/189I/343V, 68P/214R/215P/271Y/281P/282A/372L,68P/215P/271Y/343V/372L/381L, 68P/215P/377L, 68P/271Y/372L, 68P/377L,69F/108G/270E/372L/377L, 69F/189L, 69F/215K, 69F/215K/269L/270I/377L,69F/215R, 69F/285L/373G, 69L, 69L/70L/331Q/372M,69L/189E/271Y/281P/282A, 69L/189I, 69L/189I/214R/271Y/281P/282A/343V,69L/189I/271Y/343V/381L, 69L/189I/280G/282G/343V/372L/381L,69L/189I/282A, 69L/189Q/377Y, 69L/215P/271Y/280G/281P/282C,69L/215P/271Y/282A, 69L/215P/271Y/372L, 69L/215P/343V/372L/381L,69L/215R/285P/317P, 69L/271Y, 69L/271Y/372L, 69L/282C/343V/372L,69L/372L, 69M/70A/372M, 69W, 69W/70L, 69W/70L/372M, 69W/70L/459R,69W/108S, 69W/189E/214R/343V/372L, 69W/189E/271Y/343V, 69W/189E/372L,69W/189I, 69W/189I/215P/343V, 69W/189I/271Y, 69W/189I/343V/381L,69W/189Q/372L/377Y, 69W/212A/213L/215R/280G/281P,69W/214R/215P/271Y/372L/377Y/381L, 69W/214R/271Y/282A,69W/214R/271Y/343V, 69W/215K/343V, 69W/215P, 69W/215R, 69W/215R/323Y,69W/282A, 69W/372M, 69Y/108G/281P/285P, 69Y/110A/215R/281P,69Y/189L/281P/373G, 70L, 70L/212P, 108G, 108G/189I/282A/285L/320W,108G/189L, 108G/189L/320W, 108G/189L/377I, 108G/215K, 108G/215P/377L,108G/269L/270E, 108G/270E, 108G/282A/285L/377L, 108G/285L,108G/320W/323I, 108G/377I, 108G/377L, 126C,126C/184S/213S/280G/281P/285L/320G, 126C/184S/213S/372L,126C/189I/285L/372L, 126C/215P, 126C/372L, 181L/215P, 189E/372L/377Y,189I, 189I/214R/215P/271Y/282G, 189I/215K, 189I/215P/343V/372L,189I/215R/249T/277M, 189I/270E/285L, 189I/270E/372L, 189I/280G/282A,189I/320W/377I, 189I/343V, 189I/377I, 189L, 189Q, 189Q/214R,189Q/215P/271Y/281P/282C/377Y, 189Q/343V, 189Q/343V/377Y, 189Q/381L,213S/215P/320G, 214R/215P/271Y, 214R/215P/271Y/377Y, 214R/271Y,214R/280G/282A/343V/377Y/381L, 215K, 215K/281P/285L/372L,215K/281P/373G, 215K/285L/317P, 215K/285L/445L, 215K/323Y, 215K/372L,215K/372L/377L, 215K/373G, 215P, 215P/271Y/372L, 215P/320G,215P/320G/372L, 215P/372L, 215P/372L/377L, 215P/377L, 215P/381L, 215R,215R/249T/280G/281P/285L/372L, 215R/280G/281P/285L/372L,215R/281P/285L/373G, 215R/285P, 215R/320G, 215R/372L, 215W,215W/285L/346S, 215W/285P, 215W/373G, 249T/377L,269L/270E/281P/372L/377L, 270E/377L, 271Y, 271Y/343V, 271Y/343V/372L,271Y/343V/372L/381L, 280G/285L/372L, 281P/372L, 282A/285L/320W/323I,285L/323I, 320W, 343V/372L, 372L, 372L/377L, 372L/381L, 372M, 373G, and377L, wherein the amino acid positions are numbered with reference toSEQ ID NO: 8. In some further embodiments, the polynucleotide sequenceencodes an engineered carboxyesterase comprisng at least onesubstitution or substitution set selected from: P63A, P63A/M189A,P63A/N215R/A343V, P63R, P63R/Y65G/A108G, P63R/Y65G/A108G/M189L,P63R/Y65G/A108G/F377I, P63R/Y65G/T282A/A285L/Y320W/F323I,P63R/Y65G/Y320W/F323I, P63R/A108G, P63R/A108G/T282A/A285L/F377L,P63R/A108G/A285L/F377I, P63R/A108G/Y320W/F323C, P63R/F377I, P63T/N215R,P63Y, P63Y/M189L, P63Y/G212P/N215R, P63Y/G212P/N215R/P268A/A269N/A343V,P63Y/N215P/A269N, P63Y/N215R, P63Y/N215R/V270I/W271S,P63Y/P268A/A269N/V270I/A429V, Y65G/Y320W, Y65G/Y320W/F323I,Y65W/I69L/I372L, Y65W/I69M/G70A/L281P/I372L, Y65W/I69W/G70L/I372L,Y65W/G70L/I372M, A68P, A68P/I69L/M189E/G214R/W271Y/V280G,A68P/I69L/M189E/G214R/I372L, A68P/I69L/M189I/G214R/N215P/W271Y,A68P/I69L/M189I/L281P/T282C/I372L/F377Y/A381L, A68P/I69L/M189Q/G214R,A68P/I69L/M189Q/W271Y/V280G/I372L/A381L, A68P/I69L/N215P,A68P/I69L/W271Y, A68P/I69L/T282C/V287I, A68P/I69L/A343V/I372L,A68P/I69W/M189E/G214R/N215P/W271Y/L281P/T282G/A343V/A381L,A68P/I69W/M189E/V280G/L281P/T282A/I372L/F377Y,A68P/I69W/M189E/A343V/A381L, A68P/I69W/M189I/G214R/N215P,A68P/I69W/M189I/G214R/F377Y/A381L, A68P/I69W/M189I/W271Y,A68P/I69W/M189I/I372L, A68P/I69W/M189I/A381L,A68P/I69W/G214R/N215P/W271Y, A68P/I69W/G214R/A343V, A68P/I69W/N215P,A68P/A108G/F377L, A68P/E184S, A68P/E184S/M189E, A68P/M189I/W271Y/I372L,A68P/M189I/A343V, A68P/G214R/N215P/W271Y/L281P/T282A/I372L,A68P/N215P/W271Y/A343V/I372L/A381L, A68P/N215P/F377L, A68P/W271Y/I372L,A68P/F377L, I69F/A108G/V270E/I372L/F377L, I69F/M189L, I69F/N215K,I69F/N215K/A269L/V270I/F377L, I69F/N215R, I69F/A285L/T373G, I69L,I69L/G70L/P331Q/I372M, I69L/M189E/W271Y/L281P/T282A, I69L/M189I,I69L/M189I/G214R/W271Y/L281P/T282A/A343V, I69L/M189I/W271Y/A343V/A381L,I69L/M189I/V280G/T282G/A343V/I372L/A381L, I69L/M189I/T282A,I69L/M189Q/F377Y, I69L/N215P/W271Y/V280G/L281P/T282C,I69L/N215P/W271Y/T282A, I69L/N215P/W271Y/I372L,I69L/N215P/A343V/I372L/A381L, I69L/N215R/A285P/T317P, I69L/W271Y,I69L/W271Y/I372L, I69L/T282C/A343V/I372L, I69L/I372L, I69M/G70A/I372M,I69W, I69W/G70L, I69W/G70L/I372M, I69W/G70L/G459R, I69W/A108S,I69W/M189E/G214R/A343V/I372L, I69W/M189E/W271Y/A343V, I69W/M189E/I372L,I69W/M189I, I69W/M189I/N215P/A343V, I69W/M189I/W271Y,I69W/M189I/A343V/A381L, I69W/M189Q/I372L/F377Y,I69W/G212A/A213L/N215R/V280G/L281P,I69W/G214R/N215P/W271Y/I372L/F377Y/A381L, I69W/G214R/W271Y/T282A,I69W/G214R/W271Y/A343V, I69W/N215K/A343V, I69W/N215P, I69W/N215R,I69W/N215R/F323Y, I69W/T282A, I69W/I372M, I69Y/A108G/L281P/A285P,I69Y/T110A/N215R/L281P, I69Y/M189L/L281P/T373G, G70L, G70L/G212P, A108G,A108G/M189I/T282A/A285L/Y320W, A108G/M189L, A108G/M189L/Y320W,A108G/M189L/F377I, A108G/N215K, A108G/N215P/F377L, A108G/A269L/V270E,A108G/V270E, A108G/T282A/A285L/F377L, A108G/A285L, A108G/Y320W/F323I,A108G/F377I, A108G/F377L, R126C,R126C/E184S/A213S/V280G/L281P/A285L/Y320G, R126C/E184S/A213S/I372L,R126C/M189I/A285L/I372L, R126C/N215P, R126C/I372L, V181L/N215P,M189E/I372L/F377Y, M189I, M189I/G214R/N215P/W271Y/T282G, M189I/N215K,M189I/N215P/A343V/I372L, M189I/N215R/A249T/G277M, M189I/V270E/A285L,M189I/V270E/I372L, M189I/V280G/T282A, M189I/Y320W/F377I, M189I/A343V,M189I/F377I, M189L, M189Q, M189Q/G214R,M189Q/N215P/W271Y/L281P/T282C/F377Y, M189Q/A343V, M189Q/A343V/F377Y,M189Q/A381L, A213S/N215P/Y320G, G214R/N215P/W271Y,G214R/N215P/W271Y/F377Y, G214R/W271Y,G214R/V280G/T282A/A343V/F377Y/A381L, N215K, N215K/L281P/A285L/I372L,N215K/L281P/T373G, N215K/A285L/T317P, N215K/A285L/V445L, N215K/F323Y,N215K/I372L, N215K/I372L/F377L, N215K/T373G, N215P, N215P/W271Y/I372L,N215P/Y320G, N215P/Y320G/I372L, N215P/I372L, N215P/I372L/F377L,N215P/F377L, N215P/A381L, N215R, N215R/A249T/V280G/L281P/A285L/I372L,N215R/V280G/L281P/A285L/I372L, N215R/L281P/A285L/T373G, N215R/A285P,N215R/Y320G, N215R/I372L, N215W, N215W/A285L/G346S, N215W/A285P,N215W/T373G, A249T/F377L, A269L/V270E/L281P/I372L/F377L, V270E/F377L,W271Y, W271Y/A343V, W271Y/A343V/I372L, W271Y/A343V/I372L/A381L,V280G/A285L/I372L, L281P/I372L, T282A/A285L/Y320W/F323I, A285L/F323I,Y320W, A343V/I372L, I372L, I372L/F377L, I372L/A381L, I372M, T373G, andF377L, wherein the amino acid positions are numbered with reference toSEQ ID NO: 8.

The present invention also provides polynucleotide sequences encoding atleast one engineered carboxyesterase or a functional fragment thereof,the polynucleotide sequence comprising at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, and/or 139.

The present invention further provides polynucleotide sequences encodingengineered carboxyesterases provided herein, wherein the polynucleotidesequence is operably linked to a control sequence. In some additionalembodiments, the polynucleotide sequences are codon optimized.

The present invention also provides expression vectors comprising atleast one polynucleotide sequence encoding an engineered carboxyesteraseprovided herein. In addition, the present invention provides host cellscomprising at least one expression vector provided herein. In someembodiments, the present invention also provides host cells comprisingat least one polynucleotide sequence encoding at least one engineeredcarboxyesterase provided herein.

The present invention also provides methods of producing an engineeredcarboxyesterase in a host cell, comprising culturing a host cellcomprising an expression vector comprising at least one polynucleotideencoding at least one engineered carboxyesterase, under suitableconditions, such that at least one engineered carboxyesterase isproduced. In some embodiments, the methods further comprise recoveringat least one engineered carboxyesterase from the culture and/or hostcell. In some additional embodiments, the methods further comprise thestep of purifying the at least one engineered carboxyesterase.

DESCRIPTION OF THE INVENTION

The present invention provides engineered carboxyesterases (E.C. 3.1.1)having improved non-native properties as compared to naturally occurringwild-type (WT) carboxyesterase enzymes, as well as polynucleotidesencoding the engineered carboxyesterase enzymes, host cells capable ofexpressing the engineered carboxyesterase enzymes, and methods ofapplying the engineered carboxyesterase enzymes to amidation reactions.

Switching enzyme function and enzyme substrate scope is feasible, as ithas been observed that enzyme active sites are capable of catalyzingseveral different chemical reactions via one amino acid mutation (SeeRauwerdink and Kazluaskas, ACS Cat., 5: 6153-6176 [2015]). To improvecarboxyesterase functionality, and substrate scope, wild-typecarboxyesterases were subjected to directed evolution. The resultantvariants possessed improved capabilities in the generation of amidebonds using a diverse set of amine and carboxyester substrate pairs(See, Scheme 1, below). The engineered carboxyesterases had activity notonly in aqueous systems, but also are active in the presence of organicco-solvents and even near-total organic solvent concentrations (e.g.,˜98% v/v), as described herein. Further, immobilization of theseengineered carboxyesterases facilitates continuous flow operations foramide production, aids in the purification of the final amide product,as well as improves the efficiency and overall cost of amidationoperations.

The present invention provides novel engineered carboxyesterasepolypeptides, along with their corresponding polynucleotide sequencesand methods of application, which demonstrate general amide bondformation (See, Scheme 1, below). In some embodiments, the engineeredpolypeptides possess modified properties that broaden the functionalityand scope of activity of these enzymes as compared to the naturallyoccurring wild-type Thermobifida fusca (T. fusca) carboxyesterase (SEQID NO: 2). The improved carboxyesterase properties include, but are notlimited to: solvent stability, enzymatic activity, regiospecificity,stereoselectivity, reduced host cell toxicity, thermal stability, pHstability, substrate scope, and/or reduced substrate or productinhibition. The present invention also provides polynucleotides thathave been improved to facilitate expression of the desired polypeptidesin non-natural host organisms (e.g., E. coli).

In some embodiments, the carboxyesterase polypeptides provided hereinpossess modified properties that expand the functionality and scope ofactivity of these enzymes as compared to the naturally occurringwild-type Geobacillus stearothermophilus carboxyesterase (SEQ ID NO:138). In some instances, these polypeptides are carboxyesterase enzymeswhich are enhanced relative to the wild-type Mycobacterium tuberculosiscarboxyesterase (SEQ ID NO: 140). Further, in some embodiments, thepresent invention provides polynucleotides that have been improved tofacilitate expression of the desired polypeptides in a non-native hostorganisms (e.g., E. coli).

The improved properties of the carboxyesterase variants presented arerelated to the engineered amidation polypeptides containing residuedifferences at specific residue positions as compared to the referencecarboxyesterase sequence of T. fusca or another referred engineeredamidation polypeptide, such as the sequence of SEQ ID NO: 8. In someembodiments, the residue differences are present at least one of thefollowing amino acid positions: X39, X62, X63, X64, X65, X66, X68, X69,X70, X71, X77, X103, X104, X105, X107, X108, X109, X110, X111, X113,X114, X115, X117, X118, X119, X126, X147, X153, X164, X174, X181, X183,X184, X185, X186, X187, X188, X189, X190, X209, X210, X211, X212, X213,X214, X215, X216, X217, X224, X231, X249, X263, X268, X269, X270, X271,X276, X277, X278, X279, X280, X281, X282, X283, X284, X285, X286, X287,X311, X317, X320, X321, X323, X324, X331, X343, X345, X346, X349, X372,X373, X374, X376, X377, X381, X405, X416, X420, X427, X428, X429, X438,X445, X459, and X470.

In some embodiments, the engineered carboxyesterases provided herein arecharacterized as exhibiting increased thermostability as compared to thewild-type polypeptide under the same reaction conditions. The engineeredcarboxyesterases are capable of mediating amidation conversion (See,Scheme 1, below), as indicated by continued formation of products, athigher temperatures and for longer times than the WT carboxyesterase. Insome embodiments, the engineered carboxyesterase polypeptides maintainor have increased activity in the presence of higher concentrations ofsubstrate ester (I) and/or amine (II), such as 300 mM isobutylamine. Insome embodiments, the engineered carboxyesterase polypeptides maintainor have increased activity under conditions with various pH levels(e.g., pH 9.0), as compared to the WT carboxyesterase. In someembodiments, the engineered polypeptides with increased thermostability,pH stability, and/or substrate stability comprise and amino acidsequence that is at least 80%, 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to thereference sequence of SEQ ID NOs: 2 and/or 8.

In some embodiments, the engineered carboxyesterases are capable ofbiocatalytic activity improvements for converting the substratecompound(s) to product(s) (See, Scheme 1, below) at least about1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 150-fold, 500-fold or more relative to theactivity of wild-type carboxyesterase (SEQ ID NO: 2) or a referenceengineered carboxyesterase (SEQ ID NO: 8), under suitable reactionconditions. In some embodiments, these improvements in enzyme activityextend to associated increases in thermostability, stereoselectivity,stereospecificity, regiospecificity, solvent stability, pH stability,and/or substrate binding, or reduced substrate and/or productinhibition.

In some embodiments, the engineered carboxyesterases are characterizedby activity on a variety of structurally different carboxyester (I) oramine (II) substrates. In some embodiments, engineered polypeptides arecapable of biocatalytically converting esters (N,N-diethylamino glycinemethyl ester, ethyl benzoate, ethyl acetate, pyrazine-2-carboxylic ethylester, 1H-indole-4-carboxylic ethyl ester, methyl phenylacetate), andamines (n-butylamine, isobutylamine, aniline, benzylamine,2,6-dimethylaniline, t-butylamine, N′-t-butoxycarbonyl-benzhydrazide,4-methylpiperidine, 0-t-butylhydroxylamine, 2,6-dimethylaniline, orstereoselective conversion of (S)-(−)-α-methylbenzylamine,(R)-(+)-α-methylbenzylamine), to their corresponding amide product at agreater rate than the WT polypeptides of SEQ ID NO: 2 and/or theengineered polypeptide SEQ ID NO: 8.

In some embodiments, the improved engineered variant polypeptidecomprises an amino acid sequence corresponding to SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88. 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, or 140.In some embodiments, the carboxyesterase enzymes provided herein areobtained by mutagenizing a gene encoding an engineered carboxyesterasepolypeptide that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the aminoacid sequence of the naturally-occurring T. fusca carboxyesterase (SEQID NO: 2).

In some additional embodiments of the present invention, thecarboxyesterase polypeptide variants are encoded by polynucleotides orpolynucleotides that hybridize to yield such polynucleotides underhighly stringent conditions, as provided herein. In some embodiments,the polynucleotides comprise promoters and/or other regulatory elementsuseful for expression of the encoded engineered carboxyesterase, and canutilize codons optimized for specific expression systems.

In some embodiments, the polynucleotides encoding the improvedcarboxyesterase enzymes comprise a sequence selected from SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,and 139.

In some additional embodiments, the present invention provides hostcells comprising the polynucleotides and/or expression vectors providedherein. In some embodiments, the host cells are T. fusca, while in somealternative embodiments, they are other organisms (e.g., E. coli). Thehost cells find use in the expression of the encoded polynucleotides toproduce the engineered carboxyesterases, and isolation of the engineeredcarboxyesterases described herein. In some embodiments, the host cellsfine use in directly converting substrate(s) to the desired product(s).

In some additional embodiments, the present invention provides methodsfor carrying out reaction Scheme 1 (shown below) using any of theengineered carboxyesterase enzymes provided herein. In some embodiments,the methods comprise contacting or incubating carboxyester (I) and amine(II) substrates with an engineered carboxyesterase polypeptide of thepresent invention under suitable reaction conditions for the conversionof the substrates to the corresponding amide product, therebytransforming the substrates to the product compounds. Whether carryingout the method with whole cells, cell extracts or purifiedcarboxyesterase enzymes, a single carboxyesterase enzyme can be used or,alternatively, mixtures of at least two carboxyesterase enzymes finduse.

The engineered carboxyesterases of the present invention are capable ofconverting a diverse set of carboxyester (Scheme 1, I) and amine (II)substrates to their corresponding amide products (III). The scope ofcarboxyester substrates available can be detailed while considering R¹and R² of formula (I). In some embodiments, the functionality of groupsat R¹ and R² encompasses various components, from a hydrogen atom toalkyl, alkenyl, alkynyl, alkoxy, carboxy, heteroalkyl, heteroalkenyl,heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkythioalkyl,cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, andheteroarylalkyl components, excepting that R² cannot be a hydrogen. Insome additional embodiments, selected functionality at the R³ and R⁴positions of the amine (II) consist of a hydrogen atom, alkyl, alkenyl,alkynyl, alkoxy, carboxy, heteroalkyl, heteroalkenyl, heteroalkynyl,carboxyalkyl, aminoalkyl, haloalkyl, alkythioalkyl, cycloalkyl, aryl,arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl. In somefurther embodiments, the selected R¹ and R² may be linked to form a3-membered to 10-membered ring, with the caveat that groups at R³ and R⁴are separately chosen from alkyl, alkenyl, alkynyl, alkoxy, carboxy,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkythioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl,heteroaryl, heteroarylalkyl, and may also be linked to form a 3-memberedto 10-membered ring; and optionally R′ or R² may be linked via a alkyl,alkenyl, alkynyl, alkoxy, carboxy, heteroalkyl, heteroalkenyl,heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkythioalkyl,cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, orheteroarylalkyl tether to R³ or R⁴. The amidation process proceeds asthe compound (I),

wherein R¹, and R² are as defined above, and a compound of formula (II),

wherein R³, and R⁴ are as defined above, and an engineered polypeptidehaving amidative activity under suitable reaction conditions. In someembodiments of the reaction methods provided herein, the WT T. fuscacarboxyesterase (SEQ ID NO: 2), or a reference engineered polypeptide(e.g., SEQ ID NO: 8), carboxyesterase derivatives presented herein arecapable of generating primary and secondary amides matching the genericformula (III). The engineered amidation enzymes provided herein (e.g.,the engineered carboxyesterase polypeptides of even numbered sequenceidentifiers SEQ ID NO: 4-136) find use as biocatalysts of the reactionabove (Scheme 1).

Definitions

In reference to the present invention, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings. All U.S patents and published U.S. patentapplications, including all sequences disclosed within such patents andpatent applications, referred to herein are expressly incorporated byreference. Unless otherwise indicated, the practice of the presentinvention involves conventional techniques commonly used in molecularbiology, fermentation, microbiology, and related fields, which are knownto those of skill in the art. Unless defined otherwise herein, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, the preferred methods and materialsare described. Indeed, it is intended that the present invention not belimited to the particular methodology, protocols, and reagents describedherein, as these may vary, depending upon the context in which they areused. The headings provided herein are not limitations of the variousaspects or embodiments of the present invention.

Nonetheless, in order to facilitate understanding of the presentinvention, a number of terms are defined below. Numeric ranges areinclusive of the numbers defining the range. Thus, every numerical rangedisclosed herein is intended to encompass every narrower numerical rangethat falls within such broader numerical range, as if such narrowernumerical ranges were all expressly written herein. It is also intendedthat every maximum (or minimum) numerical limitation disclosed hereinincludes every lower (or higher) numerical limitation, as if such lower(or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and“the” include the plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “host cell” includes aplurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation and amino acid sequences are written left to rightin amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention that can be had by reference to thespecification as a whole. Accordingly, the terms defined below are morefully defined by reference to the specification as a whole.

As used herein, “carboxyesterases” are defined as enzymes that naturallyhave catalytic activity toward the hydrolysis of carboxyesters whichresults in the formation of an organic acid and an alcohol.

As used herein, “amidation,” or amide synthesis, refers to the processof generating an amide bond, resulting in a carboxamide (organic amide).

As used herein, the terms “protein,” “polypeptide,” and “peptide” areused interchangeably herein to denote a polymer of at least two aminoacids covalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids.

As used herein, “polynucleotide” and “nucleic acid” refer to two or morenucleosides that are covalently linked together. The polynucleotide maybe wholly comprised ribonucleosides (i.e., an RNA), wholly comprised of2′ deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), itmay include one or more modified and/or synthetic nucleobases (e.g.,inosine, xanthine, hypoxanthine, etc.). In one embodiment of theinvention, such modified or synthetic nucleobases will be encodingnucleobases.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, “naturally occurring,” “wild-type,” and “WT” refer tothe form found in nature. For example, a naturally occurring orwild-type polypeptide or polynucleotide sequence is a sequence presentin an organism that can be isolated from a source in nature and whichhas not been intentionally modified by human manipulation.

As used herein, “non-naturally occurring” or “engineered” or“recombinant” when used in the present invention with reference to(e.g., a cell, nucleic acid, or polypeptide), refers to a material, or amaterial corresponding to the natural or native form of the material,that has been modified in a manner that would not otherwise exist innature, or is identical thereto but produced or derived from syntheticmaterials and/or by manipulation using recombinant techniques.Non-limiting examples include, among others, recombinant cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

As used herein, “percentage of sequence identity,” “percent identity,”and “percent identical” refer to comparisons between polynucleotidesequences or polypeptide sequences, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whicheither the identical nucleic acid base or amino acid residue occurs inboth sequences or a nucleic acid base or amino acid residue is alignedwith a gap to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Determination of optimal alignment and percentsequence identity is performed using the BLAST and BLAST 2.0 algorithms(See, e.g., Altschul et al., J. Mol. Biol. 215: 403-410 [1990]; andAltschul, et al., Nucleic Acids Res. 3389-3402 [1977]). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length within thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as, the neighborhood word scorethreshold (Altschul, et al., supra). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (See, e.g., Henikoff and Henikoff, Proc Natl Acad Sci USA89:10915 [1989]).

Numerous other algorithms are available and known in the art thatfunction similarly to BLAST in providing percent identity for twosequences. Optimal alignment of sequences for comparison can beconducted using any suitable method known in the art (e.g., by the localhomology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 [1981];by the homology alignment algorithm of Needleman and Wunsch, J. Mol.Biol. 48:443 [1970]; by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; and/or bycomputerized implementations of these algorithms [GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package]), or by visualinspection, using methods commonly known in the art. Additionally,determination of sequence alignment and percent sequence identity canemploy the BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using the default parameters provided.

As used herein, “reference sequence” refers to a defined sequence towhich another sequence is compared. A reference sequence may be a subsetof a larger sequence, for example, a segment of a full-length gene orpolypeptide sequence. Generally, a reference sequence is at least 20nucleotide or amino acid residues in length, at least 25 residues inlength, at least 50 residues in length, or the full length of thenucleic acid or polypeptide. Since two polynucleotides or polypeptidesmay each (1) comprise a sequence (i.e., a portion of the completesequence) that is similar between the two sequences, and (2) may furthercomprise a sequence that is divergent between the two sequences,sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides over a comparison window to identify and compare localregions of sequence similarity. The term “reference sequence” is notintended to be limited to wild-type sequences, and can includeengineered or altered sequences. For example, in some embodiments, a“reference sequence” can be a previously engineered or altered aminoacid sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acids residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to”, “reference to” or “relative to” whenused in the context of the numbering of a given amino acid orpolynucleotide sequence refers to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered carboxyesterase, can be aligned to a reference sequence byintroducing gaps to optimize residue matches between the two sequences.In these cases, although the gaps are present, the numbering of theresidue in the given amino acid or polynucleotide sequence is made withrespect to the reference sequence to which it has been aligned. As usedherein, a reference to a residue position, such as “Xn” as furtherdescribed below, is to be construed as referring to “a residuecorresponding to”, unless specifically denoted otherwise. Thus, forexample, “X94” refers to any amino acid at position 94 in a polypeptidesequence (e.g., SEQ ID NOs:2, 4, 10, 26, or 42).

As used herein, “stereoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one stereoisomer over anotherstereoisomer or another set of stereoisomers. Stereoselectivity can bepartial, where the formation of a stereoisomer is favored over another,or it may be complete where only one stereoisomer is formed. When thestereoisomers are enantiomers, the stereoselectivity is referred to asenantioselectivity, the fraction (typically reported as a percentage) ofone enantiomer in the sum of both enantiomers. It is commonlyalternatively reported in the art (typically as a percentage) as theenantiomeric excess (e.e.) calculated therefrom according to the formula[major enantiomer−minor enantiomer]/[major enantiomer+minor enantiomer].Where the stereoisomers are diastereoisomers, the stereoselectivity isreferred to as diastereoselectivity, the fraction (typically reported asa percentage) of one diastereomer in a mixture of two diastereomers,commonly alternatively reported as the diastereomeric excess (d.e.).Enantiomeric excess and diastereomeric excess are types of stereomericexcess. It is also to be understood that stereoselectivity is notlimited to single stereoisomers and can be described for sets ofstereoisomers.

As used herein, “highly stereoselective” refers to a chemical orenzymatic reaction that is capable of converting a substrate to itscorresponding chiral amide product, with at least about 75% stereomericexcess.

As used herein, “increased enzymatic activity” and “increased activity”refer to an improved property of an engineered enzyme, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of carboxyesterase) as compared to a reference enzyme.Exemplary methods to determine enzyme activity are provided in theExamples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of Km, Vmax or kcat, changesof which can lead to increased enzymatic activity. The carboxyesteraseactivity can be measured by any one of standard assays used formeasuring carboxyesterases, such as change in substrate or productconcentration. Comparisons of enzyme activities are made using a definedpreparation of enzyme, a defined assay under a set condition, and one ormore defined substrates, as further described in detail herein.Generally, when enzymes in cell lysates are compared, the numbers ofcells and the amount of protein assayed are determined as well as use ofidentical expression systems and identical host cells to minimizevariations in amount of enzyme produced by the host cells and present inthe lysates.

As used herein, “conversion” refers to the enzymatic transformation of asubstrate to the corresponding product.

As used herein “percent conversion” refers to the percent of thesubstrate that is converted to the product within a period of time underspecified conditions. Thus, for example, the “enzymatic activity” or“activity” of a carboxyesterase polypeptide can be expressed as “percentconversion” of the substrate to the product.

As used herein, “regiospecificity” refers to chemical reactions in whichone structural isomer is produced exclusively when other isomers arealso theoretically possible.

As used herein, “thermostable” or “thermal stable” are usedinterchangeably to refer to a polypeptide that is resistant toinactivation when exposed to a set of temperature conditions (e.g.,40-80° C.) for a period of time (e.g., 0.5-24 hrs) compared to theuntreated enzyme, thus retaining a certain level of residual activity(e.g., more than 60% to 80% for example) after exposure to elevatedtemperatures.

As used herein, “solvent stable” refers to the ability of a polypeptideto maintain similar activity (e.g., more than e.g., 60% to 80%) afterexposure to varying concentrations (e.g., 5-99%) of solvent (e.g.,isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone,toluene, butylacetate, methyl tert-butylether, etc.) for a period oftime (e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “amino acid difference” or “residue difference” refersto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. The positions of amino acid differencesgenerally are referred to herein as “Xn”, where n refers to thecorresponding position in the reference sequence upon which the residuedifference is based. For example, a “residue difference at position X40as compared to SEQ ID NO:2” refers to a difference of the amino acidresidue at the polypeptide position corresponding to position 40 of SEQID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has ahistidine at position 40, then a “residue difference at position X40 ascompared to SEQ ID NO:2” refers to an amino acid substitution of anyresidue other than histidine at the position of the polypeptidecorresponding to position 40 of SEQ ID NO:2. In most instances herein,the specific amino acid residue difference at a position is indicated as“XnY” where “Xn” specified the corresponding position as describedabove, and “Y” is the single letter identifier of the amino acid foundin the engineered polypeptide (i.e., the different residue than in thereference polypeptide). In some instances, the present invention alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present invention can include atleast one amino acid residue differences relative to a referencesequence, which is indicated by a list of the specified positions whereresidue differences are present relative to the reference sequence. Insome embodiments, where more than one amino acid can be used in aspecific residue position of a polypeptide, the various amino acidresidues that can be used are separated by a “/” (e.g., X192A/G). Thepresent invention includes engineered polypeptide sequences comprisingat least one amino acid differences that include either/or bothconservative and non-conservative amino acid substitutions. The aminoacid sequences of the specific recombinant carbonic anhydrasepolypeptides included in the Sequence Listing of the present inventioninclude an initiating methionine (M) residue (i.e., M represents residueposition 1). The skilled artisan, however, understands that thisinitiating methionine residue can be removed by biological processingmachinery, such as in a host cell or in vitro translation system, togenerate a mature protein lacking the initiating methionine residue, butotherwise retaining the enzyme's properties. Consequently, the term“amino acid residue difference relative to SEQ ID NO:2 at position Xn”as used herein may refer to position “Xn” or to the correspondingposition (e.g., position (X-1)n) in a reference sequence that has beenprocessed so as to lack the starting methionine.

As used herein, the phrase “conservative amino acid substitutions”refers to the interchangeability of residues having similar side chains,and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with a hydroxyl side chain is substituted with anotheramino acid with a hydroxyl side chain (e.g., serine and threonine); anamino acids having aromatic side chains is substituted with anotheramino acid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basic side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively. The appropriate classification of any amino acid orresidue will be apparent to those of skill in the art, especially inlight of the detailed invention provided herein. Exemplary conservativesubstitutions are provided in Table 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Residue PossibleConservative Substitutions A, L, V, I Other aliphatic (A, L, V, I) Othernon-polar (A, L, V, I, G, M) G, M Other non-polar (A, L, V, I, G, M) D,E Other acidic (D, E) K, R Other basic (K, R) P none N, Q, S, T Otherpolar H, Y, W, F Other aromatic (H, Y, W, F) C none

As used herein, the phrase “non-conservative substitution” refers tosubstitution of an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification of the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thepolypeptide while retaining enzymatic activity and/or retaining theimproved properties of an engineered enzyme. Deletions can be directedto the internal portions and/or terminal portions of the polypeptide. Invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

As used herein, “insertion” refers to modification of the polypeptide byaddition of one or more amino acids to the reference polypeptide. Insome embodiments, the improved engineered carboxyesterase enzymescomprise insertions of one or more amino acids to the naturallyoccurring carboxyesterase polypeptide as well as insertions of one ormore amino acids to engineered carboxyesterase polypeptides. Insertionscan be in the internal portions of the polypeptide, or to the carboxy oramino terminus. Insertions as used herein include fusion proteins as isknown in the art. The insertion can be a contiguous segment of aminoacids or separated by one or more of the amino acids in the naturallyoccurring polypeptide.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions in a polypeptide sequence, as comparedto a reference sequence. A substitution set can have 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In someembodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant carboxyesterasesincluded in the Tables provided in the Examples.

As used herein, “fragment” refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can typically have about 80%, about 90%, about 95%,about 98%, or about 99% of the full-length carboxyesterase polypeptide,for example, the polypeptide of SEQ ID NO:4. In some embodiments, thefragment is “biologically active” (i.e., it exhibits the same enzymaticactivity as the full-length sequence).

A “functional fragment”, or a “biologically active fragment”, usedinterchangeably, herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full-length engineered T. fusca enzyme of the presentinvention) and that retains substantially all of the activity of thefull-length polypeptide.

As used herein, “isolated polypeptide” refers to a polypeptide which issubstantially separated from other contaminants that naturally accompanyit (e.g., protein, lipids, and polynucleotides). The term embracespolypeptides which have been removed or purified from theirnaturally-occurring environment or expression system (e.g., host cell orin vitro synthesis). The improved carboxyesterase enzymes may be presentwithin a cell, present in the cellular medium, or prepared in variousforms, such as lysates or isolated preparations. As such, in someembodiments, the engineered carboxyesterase polypeptides of the presentinvention can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” refers to a compositionin which the polypeptide species is the predominant species present(i.e., on a molar or weight basis it is more abundant than any otherindividual macromolecular species in the composition), and is generallya substantially purified composition when the object species comprisesat least about 50 percent of the macromolecular species present by moleor % weight. Generally, a substantially pure engineered carboxyesterasepolypeptide composition will comprise about 60% or more, about 70% ormore, about 80% or more, about 90% or more, about 91% or more, about 92%or more, about 93% or more, about 94% or more, about 95% or more, about96% or more, about 97% or more, about 98% or more, or about 99% of allmacromolecular species by mole or % weight present in the composition.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated improved carboxyesterase polypeptide is a substantiallypure polypeptide composition.

As used herein, when used with reference to a nucleic acid orpolypeptide, the term “heterologous” refers to a sequence that is notnormally expressed and secreted by an organism (e.g., a wild-typeorganism). In some embodiments, the term encompasses a sequence thatcomprises two or more subsequences which are not found in the samerelationship to each other as normally found in nature, or isrecombinantly engineered so that its level of expression, or physicalrelationship to other nucleic acids or other molecules in a cell, orstructure, is not normally found in nature. For instance, a heterologousnucleic acid is typically recombinantly produced, having two or moresequences from unrelated genes arranged in a manner not found in nature(e.g., a nucleic acid open reading frame (ORF) of the inventionoperatively linked to a promoter sequence inserted into an expressioncassette, such as a vector). In some embodiments, “heterologouspolynucleotide” refers to any polynucleotide that is introduced into ahost cell by laboratory techniques, and includes polynucleotides thatare removed from a host cell, subjected to laboratory manipulation, andthen reintroduced into a host cell.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. In some embodiments, thepolynucleotides encoding the carboxyesterase enzymes may be codonoptimized for optimal production from the host organism selected forexpression.

As used herein, “control sequence” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide and/or polypeptide of the present invention. Each controlsequence may be native or foreign to the polynucleotide of interest.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator.

As used herein, “operably linked” is defined herein as a configurationin which a control sequence is appropriately placed (i.e., in afunctional relationship) at a position relative to a polynucleotide ofinterest such that the control sequence directs or regulates theexpression of the polynucleotide and/or polypeptide of interest.

As used herein, “suitable reaction conditions” refer to those conditionsin the biocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, temperature, pH, buffers, co-solvents, etc.) underwhich an carboxyesterase polypeptide of the present invention is capableof converting a substrate compound to a product compound (e.g.,conversion of one compound to another compound). Exemplary “suitablereaction conditions” are provided in the present invention andillustrated by the Examples.

As used herein, “loading,” such as in “compound loading,” “enzymeloading,” or “substrate loading” refers to the concentration or amountof a component in a reaction mixture at the start of the reaction.

As used herein, “substrate” in the context of a biocatalyst mediatedprocess refers to the compound or molecule acted on by the biocatalyst.

As used herein “product” in the context of a biocatalyst mediatedprocess refers to the compound or molecule resulting from the action ofthe biocatalyst.

As used herein, “equilibration” as used herein refers to the processresulting in a steady state concentration of chemical species in achemical or enzymatic reaction (e.g., interconversion of two species Aand B), including interconversion of stereoisomers, as determined by theforward rate constant and the reverse rate constant of the chemical orenzymatic reaction.

As used herein, “alkyl” refers to saturated hydrocarbon groups of from 1to 18 carbon atoms inclusively, either straight chained or branched,more preferably from 1 to 8 carbon atoms inclusively, and mostpreferably 1 to 6 carbon atoms inclusively. An alkyl with a specifiednumber of carbon atoms is denoted in parenthesis (e.g., (C1-C4) alkylrefers to an alkyl of 1 to 4 carbon atoms).

As used herein, “alkenyl” refers to groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one doublebond but optionally containing more than one double bond.

As used herein, “alkynyl” refers to groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one triplebond but optionally containing more than one triple bond, andadditionally optionally containing one or more double bonded moieties.

As used herein, “heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” referto alkyl, alkenyl and alkynyl as defined herein in which one or more ofthe carbon atoms are each independently replaced with the same ordifferent heteroatoms or heteroatomic groups. Heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NRα—, —PH—, —S(O)—, —S(O)2-, —S(O)NRα-, —S(O)2NRα-, and the like, including combinations thereof, whereeach Rα is independently selected from hydrogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

As used herein, “alkoxy” refers to the group —ORO wherein Rβ is an alkylgroup is as defined above including optionally substituted alkyl groupsas also defined herein.

As used herein, “aryl” refers to an unsaturated aromatic carbocyclicgroup of from 6 to 12 carbon atoms inclusively having a single ring(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl).Exemplary aryls include phenyl, pyridyl, naphthyl and the like.

As used herein, “amino” refers to the group —NH2. Substituted aminorefers to the group —NHRδ, NRδRδ, and NRδRδRδ, where each Rδ isindependently selected from substituted or unsubstituted alkyl,cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl,acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like.Typical amino groups include, but are limited to, dimethylamino,diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino,furanyl-oxy-sulfamino, and the like.

As used herein, “oxo” refers to ═O.

As used herein, “oxy” refers to a divalent group —O—, which may havevarious substituents to form different oxy groups, including ethers andesters.

As used herein, “carboxy” refers to —COOH.

As used herein, “carbonyl” refers to —C(O)—, which may have a variety ofsubstituents to form different carbonyl groups including acids, acidhalides, aldehydes, amides, esters, and ketones.

As used herein, “alkyloxycarbonyl” refers to —C(O)ORE, where RE is analkyl group as defined herein, which can be optionally substituted.

As used herein, “aminocarbonyl” refers to —C(O)NH2. Substitutedaminocarbonyl refers to —C(O)NRδRδ, where the amino group NRδRδ is asdefined herein.

As used herein, “halogen” and “halo” refer to fluoro, chloro, bromo andiodo.

As used herein, “hydroxy” refers to —OH.

As used herein, “cyano” refers to —CN.

As used herein, “heteroaryl” refers to an aromatic heterocyclic group offrom 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusivelyselected from oxygen, nitrogen and sulfur within the ring. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl).

As used herein, “heteroarylalkyl” refers to an alkyl substituted with aheteroaryl (i.e., heteroaryl-alkyl-groups), preferably having from 1 to6 carbon atoms inclusively in the alkyl moiety and from 5 to 12 ringatoms inclusively in the heteroaryl moiety. Such heteroarylalkyl groupsare exemplified by pyridylmethyl and the like.

As used herein, “heteroarylalkenyl” refers to an alkenyl substitutedwith a heteroaryl (i.e., heteroaryl-alkenyl-groups), preferably havingfrom 2 to 6 carbon atoms inclusively in the alkenyl moiety and from 5 to12 ring atoms inclusively in the heteroaryl moiety.

As used herein, “heteroarylalkynyl” refers to an alkynyl substitutedwith a heteroaryl (i.e., heteroaryl-alkynyl-groups), preferably havingfrom 2 to 6 carbon atoms inclusively in the alkynyl moiety and from 5 to12 ring atoms inclusively in the heteroaryl moiety.

As used herein, “heterocycle,” “heterocyclic,” and interchangeably“heterocycloalkyl,” refer to a saturated or unsaturated group having asingle ring or multiple condensed rings, from 2 to 10 carbon ring atomsinclusively and from 1 to 4 hetero ring atoms inclusively selected fromnitrogen, sulfur or oxygen within the ring. Such heterocyclic groups canhave a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiplecondensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl).Examples of heterocycles include, but are not limited to, furan,thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine,indoline and the like.

As used herein, “membered ring” is meant to embrace any cyclicstructure. The number preceding the term “membered” denotes the numberof skeletal atoms that constitute the ring. Thus, for example,cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings andcyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

Unless otherwise specified, positions occupied by hydrogen in theforegoing groups can be further substituted with substituentsexemplified by, but not limited to, hydroxy, oxo, nitro, methoxy,ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy,fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl,alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy,alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl,alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido,cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl,acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substitutedaryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl,heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl,substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl,morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; andpreferred heteroatoms are oxygen, nitrogen, and sulfur. It is understoodthat where open valences exist on these substituents they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further understood that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present invention, and is otherwise chemically reasonable.

As used herein, “optional” and “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. One of ordinary skill in the artwould understand that with respect to any molecule described ascontaining one or more optional substituents, only sterically practicaland/or synthetically feasible compounds are meant to be included.

As used herein, “optionally substituted” refers to all subsequentmodifiers in a term or series of chemical groups. For example, in theterm “optionally substituted arylalkyl, the “alkyl” portion and the“aryl” portion of the molecule may or may not be substituted, and forthe series “optionally substituted alkyl, cycloalkyl, aryl andheteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups,independently of the others, may or may not be substituted.

As used herein, “protecting group” refers to a group of atoms that mask,reduce or prevent the reactivity of the functional group when attachedto a reactive functional group in a molecule. Typically, a protectinggroup may be selectively removed as desired during the course of asynthesis. Examples of protecting groups are well-known in the art.Functional groups that can have a protecting group include, but are notlimited to, hydroxy, amino, and carboxy groups. Representative aminoprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“SES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated (e.g., methyl and ethyl esters, acetate or propionategroups or glycol esters) or alkylated such as benzyl and trityl ethers,as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers(e.g., TMS or TIPPS groups) and allyl ethers. Other protecting groupscan be found in the references noted herein.

Engineered Carboxyesterase Polypeptides

The present invention provides engineered polypeptides havingcarboxyesterase activity (also referred to herein as “engineeredcarboxyesterase polypeptides”) useful for amidation reactions.Accordingly, in one aspect, the present invention provides engineeredpolypeptides having carboxyesterase activity which are capable ofconverting substrate compound(s) to product compound(s) as shown inTable 3.1 in Example 3. Further, the present invention providespolynucleotides encoding the engineered polypeptides, associated vectorsand host cells comprising the polynucleotides, methods for making theengineered polypeptides, and methods for using the engineeredpolypeptides, including suitable reaction conditions.

The engineered polypeptides of the present invention are non-naturallyoccurring carboxyesterases engineered to have improved enzyme properties(e.g., increased stereoselectivity) as compared to the wild-typecarboxyesterase polypeptide of T. fusca (GenBank Acc. No.WP_011292850.1; SEQ ID NO: 2). In some embodiments, various engineeredcarboxyesterase polypeptides provided herein exhibit improved enzymeproperties as compared to other engineered reference carboxyesterasepolypeptides provided herein. In some embodiments, the engineeredpolypeptides of the present invention are non-naturally occurringcarboxyesterases engineered to have improved enzyme properties (e.g.,increased thermostability) as compared to the wild-type carboxyesterasepolypeptide of G. stearothermophilus (GenBank Acc. No. WP_033015113; SEQID NO: 138). In some further embodiments, the engineered polypeptidesare non-naturally occurring carboxyesterases engineered to have improvedenzyme properties (e.g., increased thermostability) as compared to thewild-type carboxyesterase polypeptide of M. tuberculosis (GenBank Acc.No. WP_003407276; SEQ ID NO: 140).

In some embodiments, the engineered carboxyesterase variants providedherein comprise polypeptide sequences having at least 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 2, a reference engineeredcarboxyesterase (SEQ ID NO: 8), or a functional fragment thereof,wherein the engineered carboxyesterase comprises at least onesubstitution or substitution set in the polypeptide sequence, andwherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some embodiments, theengineered polypeptides having carboxyesterase activity comprisepolypeptides having the amino acid substitutions provided herein (See,e.g., Tables 8.1 and 11.).

The present invention provides engineered carboxyesterases comprisingpolypeptide sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 2 or a functional fragment thereof, wherein the engineeredcarboxyesterases comprise at least one substitution or substitution setin their polypeptide sequences, and wherein the amino acid positions ofthe polypeptide sequences are numbered with reference to SEQ ID NO: 2.In some embodiments, at least one substitution or substitution set inthe polypeptide sequence comprises substitutions at positions selectedfrom: 39, 39/323, 62, 62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263,77, 77/184, 103, 103/147, 104, 104/429, 105, 107, 107/185, 108, 109,109/117, 110, 111, 113, 114, 115, 117, 118, 118/269, 118/349, 119, 126,147, 153, 153/215, 164, 164/271, 174, 174/282, 183, 184, 184/249, 185,186, 187, 188, 190, 209, 210, 211, 212, 213, 213/271, 213/345, 214, 215,215/271, 216, 217, 217/231, 224, 224/268/372, 231, 249, 249/284, 263,268, 269, 270, 270/470, 271, 271/416, 276, 277, 278, 279, 279/280/282,280, 281, 281/374, 282, 283, 283/429, 284, 284/438, 285, 286, 311, 317,320, 320/323, 320/323/372, 320/372/376, 320/376/377, 321, 323, 324, 345,349, 372, 372/376, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429,438, and 470, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 2. In some additionalembodiments, at least one substitution or substitution set in thepolypeptide sequence comprises substitutions selected from: 39/323,62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263, 77/184, 103, 103/147,104, 104/429, 105, 107, 107/185, 108, 109/117, 110, 111, 113, 114, 115,117, 118, 118/269, 118/349, 119, 126, 153, 153/215, 164/271, 174/282,183, 184, 184/249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213,213/271, 213/345, 214, 215, 215/271, 216, 217, 217/231, 224/268/372,249/284, 269, 270, 270/470, 271, 271/416, 276, 277, 278, 279,279/280/282, 280, 281, 281/374, 282, 283, 283/429, 284, 284/438, 285,286, 311, 317, 320, 320/323, 320/323/372, 320/372/376, 320/376/377, 321,323, 324, 372, 372/376, 373, 376, 377, 405, 420, 427, 428, and 429,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some additional embodiments,at least one substitution or substitution set in the polypeptidesequence comprises substitutions selected from: 39M/323I, 62H/117G, 63A,63R, 63T, 63Y, 64A, 64E, 64G, 64I, 64T, 64V, 64W, 65G, 65S, 65T, 65W,66N, 68L, 68P, 69F, 69G, 69H, 69L, 69V, 69W, 69Y, 70L, 70R, 70T, 70W,71F, 71G, 71H/263R, 71P, 71R, 71V, 71Y, 77S/184G, 103P, 103R, 103T/147S,104P, 104Q/429V, 105L, 107D/185W, 107L, 107P, 107S, 108G, 108K, 108Q,108R, 108S, 108W, 109G/117M, 110A, 110H, 110P, 110S, 111L, 111M, 111R,111S, 111V, 111W, 113P, 114A, 114H, 114Q, 115H, 115T, 115V, 117A, 117F,118G/349V, 118I, 118N, 118N/269T, 119G, 119P, 119S, 126C, 153H/215P,153L, 164R/271T, 174D/282V, 183P, 184F, 184G, 184P, 184S/249T, 184Y,185A, 185T, 186C, 186G, 186P, 186R, 186T, 187P, 188E, 188G, 190H, 190K,190L, 190M, 190Q, 190R, 190W, 209E, 209G, 209P, 209S, 209V, 210P, 210T,210W, 211I, 211L, 211R, 211V, 212A, 212P, 212R, 212S, 213C, 213E, 213L,213N, 213P, 213Q, 213R/345G, 213S, 213T/271K, 213V, 214K, 214L, 214T,214V, 215K, 215M, 215P, 215R, 215R/271R, 215W, 216P, 217G, 217L, 217P,217R, 217R/231V, 217S, 217V, 217W, 224I/268S/372F, 249V/284P, 269N,269V, 270I, 270I/470M, 270R, 271A, 271K, 271L, 271P, 271Q/416V, 271S,271T, 276F, 277M, 278H, 278S, 279C, 279E, 279G, 279L/280G/282M, 279V,280E, 280G, 280S, 281P, 281V, 281Y/374N, 282A, 282C, 282Q, 282R, 282S,282T, 282W, 283C, 283D, 283K, 283R/429V, 283T, 283V, 283Y, 284C, 284T,284T/438T, 284V, 285L, 285M, 285P, 286V, 311I, 317C, 317P, 320A, 320F,320G, 320G/323S, 320S, 320S/323S/372A, 320S/372A/376G, 320S/376G/377V,320W, 321L, 321S, 323C, 323I, 323R, 323Y, 324A, 372A/376A, 372L, 373G,376A, 376G, 376L, 376M, 377L, 377W, 377Y, 405D, 420G, 427A, 428V, and429L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some further embodiments, atleast one substitution or substitution set in the polypeptide sequencecomprises substitutions selected from: T39M/F323I, R62H/P117G, P63A,P63R, P63T, P63Y, P64A, P64E, P64G, P64I, P64T, P64V, P64W, Y65G, Y65S,Y65T, Y65W, P66N, A68L, A68P, I69F, I69G, I69H, I69L, I69V, I69W, I69Y,G70L, G70R, G70T, G70W, A71F, A71G, A71H/Q263R, A71P, A71R, A71V, A71Y,F775/E184G, W103P, W103R, W103T/P147S, I104P, I104Q/A429V, H105L,G107D/S185W, G107L, G107P, G107S, A108G, A108K, A108Q, A108R, A108S,A108W, F109G/P117M, T110A, T110H, T110P, T110S, N111L, N111M, N111R,N111S, N111V, N111W, S113P, G114A, G114H, G114Q, S115H, S115T, S115V,P117A, P117F, V118G/A349V, V118I, V118N, V118N/A269T, Y119G, Y119P,Y119S, R126C, R153H/N215P, R153L, W164R/W271T, G174D/L282V, G183P,E184F, E184G, E184P, E184S/A249T, E184Y, S185A, S185T, A186C, A186G,A186P, A186R, A186T, G187P, A188E, A188G, S190H, S190K, S190L, S190M,S190Q, S190R, S190W, L209E, L209G, L209P, L209S, L209V, Q210P, Q210T,Q210W, S211I, S211L, S211R, S211V, G212A, G212P, G212R, G212S, A213C,A213E, A213L, A213N, A213P, A213Q, A213R/S345G, A213S, A213T/W271K,A213V, G214K, G214L, G214T, G214V, N215K, N215M, N215P, N215R,N215R/W271R, N215W, M216P, A217G, A217L, A217P, A217R, A217R/A231V,A217S, A217V, A217W, T224I/P268S/I372F, A249V/F284P, A269N, A269V,V270I, V270I/V470M, V270R, W271A, W271K, W271L, W271P, W271Q/A416V,W271S, W271T, A276F, G277M, G278H, G278S, S279C, S279E, S279G,S279L/V280G/L282M, S279V, V280E, V280G, V280S, L281P, L281V,L281Y/D374N, L282A, L282C, L282Q, L282R, L282S, L282T, L282W, P283C,P283D, P283K, P283R/A429V, P283T, P283V, P283Y, F284C, F284T,F284T/P438T, F284V, A285L, A285M, A285P, P286V, L311I, T317C, T317P,Y320A, Y320F, Y320G, Y320G/F323S, Y320S, Y320S/F323S/I372A,Y320S/I372A/V376G, Y320S/V376G/F377V, Y320W, R321L, R321S, F323C, F323I,F323R, F323Y, L324A, I372A/V376A, I372L, T373G, V376A, V376G, V376L,V376M, F377L, F377W, F377Y, P405D, P420G, D427A, R428V, and A429L,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some embodiments, theengineered carboxyesterases comprise a substitution at position 282,wherein the position is numbered with reference to SEQ ID NO: 2. In somefurther embodiments, the substitution at position 282 is aliphatic,non-polar, basic, polar, or aromatic. In yet some additionalembodiments, the substitution selected from: X282T, X282G, X282A, X282V,X282M, X282C, X282W, X282Q, X282S, X282T, and X282R.

The present invention also provides engineered carboxyesterasescomprising a polypeptide sequences having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 8 or a functional fragment thereof,wherein the engineered carboxyesterases comprises at least onesubstitution or substitution set in the polypeptide sequences, whereinthe amino acid positions of the polypeptide sequences are numbered withreference to SEQ ID NO: 8. In some embodiments, at least onesubstitution or substitution set in the polypeptide sequence comprisessubstitutions at positions selected from: 63, 63/65/108, 63/65/108/189,63/65/108/377, 63/65/282/285/320/323, 63/65/320/323, 63/108,63/108/282/285/377, 63/108/285/377, 63/108/320/323, 63/189, 63/212/215,63/212/215/268/269/343, 63/215, 63/215/269, 63/215/270/271, 63/215/343,63/268/269/270/429, 63/377, 65/69/70/281/372, 65/69/70/372, 65/69/372,65/70/372, 65/320, 65/320/323, 68, 68/69/189/214, 68/69/189/214/215,68/69/189/214/215/271, 68/69/189/214/215/271/281/282/343/381,68/69/189/214/271/280, 68/69/189/214/372, 68/69/189/214/377/381,68/69/189/271, 68/69/189/271/280/372/381, 68/69/189/280/281/282/372/377,68/69/189/281/282/372/377/381, 68/69/189/343/381, 68/69/189/372,68/69/189/381, 68/69/214/215/271, 68/69/214/343, 68/69/215, 68/69/271,68/69/282/287, 68/69/343/372, 68/108/377, 68/184, 68/184/189,68/189/271/372, 68/189/343, 68/214/215/271/281/282/372,68/215/271/343/372/381, 68/215/377, 68/271/372, 68/377, 69, 69/70,69/70/331/372, 69/70/372, 69/70/459, 69/108, 69/108/270/372/377,69/108/281/285, 69/110/215/281, 69/189, 69/189/214/271/281/282/343,69/189/214/343/372, 69/189/215/343, 69/189/271, 69/189/271/281/282,69/189/271/343, 69/189/271/343/381, 69/189/280/282/343/372/381,69/189/281/373, 69/189/282, 69/189/343/381, 69/189/372, 69/189/372/377,69/189/377, 69/212/213/215/280/281, 69/214/215/271/372/377/381,69/214/271/282, 69/214/271/343, 69/215, 69/215/269/270/377,69/215/271/280/281/282, 69/215/271/282, 69/215/271/372, 69/215/285/317,69/215/323, 69/215/343, 69/215/343/372/381, 69/271, 69/271/372, 69/282,69/282/343/372, 69/285/373, 69/372, 70, 70/212, 108, 108/189,108/189/282/285/320, 108/189/320, 108/189/377, 108/215, 108/215/377,108/269/270, 108/270, 108/282/285/377, 108/285, 108/320/323, 108/377,126, 126/184/213/280/281/285/320, 126/184/213/372, 126/189/285/372,126/215, 126/372, 181/215, 189, 189/214, 189/214/215/271/282, 189/215,189/215/249/277, 189/215/271/281/282/377, 189/215/343/372, 189/270/285,189/270/372, 189/280/282, 189/320/377, 189/343, 189/343/377,189/372/377, 189/377, 189/381, 213/215/320, 214/215/271,214/215/271/377, 214/271, 214/280/282/343/377/381, 215,215/249/280/281/285/372, 215/271/372, 215/280/281/285/372,215/281/285/372, 215/281/285/373, 215/281/373, 215/285, 215/285/317,215/285/346, 215/285/445, 215/320, 215/320/372, 215/323, 215/372,215/372/377, 215/373, 215/377, 215/381, 249/377, 269/270/281/372/377,270/377, 271, 271/343, 271/343/372, 271/343/372/381, 280/285/372,281/372, 282/285/320/323, 285/323, 320, 343/372, 372, 372/377, 372/381,373, and 377, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 8. In some furtherembodiments, at least one substitution or substitution set in thepolypeptide sequence comprises substitutions selected from 63A,63A/189A, 63A/215R/343V, 63R, 63R/65G/108G, 63R/65G/108G/189L,63R/65G/108G/377I, 63R/65G/282A/285L/320W/323I, 63R/65G/320W/323I,63R/108G, 63R/108G/282A/285L/377L, 63R/108G/285L/377I,63R/108G/320W/323C, 63R/377I, 63T/215R, 63Y, 63Y/189L, 63Y/212P/215R,63Y/212P/215R/268A/269N/343V, 63Y/215P/269N, 63Y/215R,63Y/215R/270I/271S, 63Y/268A/269N/270I/429V, 65G/320W, 65G/320W/323I,65W/69L/372L, 65W/69M/70A/281P/372L, 65W/69W/70L/372L, 65W/70L/372M,68P, 68P/69L/189E/214R/271Y/280G, 68P/69L/189E/214R/372L,68P/69L/189I/214R/215P/271Y, 68P/69L/189I/281P/282C/372L/377Y/381L,68P/69L/189Q/214R, 68P/69L/189Q/271Y/280G/372L/381L, 68P/69L/215P,68P/69L/271Y, 68P/69L/282C/287I, 68P/69L/343V/372L,68P/69W/189E/214R/215P/271Y/281P/282G/343V/381L,68P/69W/189E/280G/281P/282A/372L/377Y, 68P/69W/189E/343V/381L,68P/69W/189I/214R/215P, 68P/69W/189I/214R/377Y/381L, 68P/69W/189I/271Y,68P/69W/189I/372L, 68P/69W/189I/381L, 68P/69W/214R/215P/271Y,68P/69W/214R/343V, 68P/69W/215P, 68P/108G/377L, 68P/184S, 68P/184S/189E,68P/189I/271Y/372L, 68P/189I/343V, 68P/214R/215P/271Y/281P/282A/372L,68P/215P/271Y/343V/372L/381L, 68P/215P/377L, 68P/271Y/372L, 68P/377L,69F/108G/270E/372L/377L, 69F/189L, 69F/215K, 69F/215K/269L/270I/377L,69F/215R, 69F/285L/373G, 69L, 69L/70L/331Q/372M,69L/189E/271Y/281P/282A, 69L/189I, 69L/189I/214R/271Y/281P/282A/343V,69L/189I/271Y/343V/381L, 69L/189I/280G/282G/343V/372L/381L,69L/189I/282A, 69L/189Q/377Y, 69L/215P/271Y/280G/281P/282C,69L/215P/271Y/282A, 69L/215P/271Y/372L, 69L/215P/343V/372L/381L,69L/215R/285P/317P, 69L/271Y, 69L/271Y/372L, 69L/282C/343V/372L,69L/372L, 69M/70A/372M, 69W, 69W/70L, 69W/70L/372M, 69W/70L/459R,69W/108S, 69W/189E/214R/343V/372L, 69W/189E/271Y/343V, 69W/189E/372L,69W/189I, 69W/189I/215P/343V, 69W/189I/271Y, 69W/189I/343V/381L,69W/189Q/372L/377Y, 69W/212A/213L/215R/280G/281P,69W/214R/215P/271Y/372L/377Y/381L, 69W/214R/271Y/282A,69W/214R/271Y/343V, 69W/215K/343V, 69W/215P, 69W/215R, 69W/215R/323Y,69W/282A, 69W/372M, 69Y/108G/281P/285P, 69Y/110A/215R/281P,69Y/189L/281P/373G, 70L, 70L/212P, 108G, 108G/189I/282A/285L/320W,108G/189L, 108G/189L/320W, 108G/189L/377I, 108G/215K, 108G/215P/377L,108G/269L/270E, 108G/270E, 108G/282A/285L/377L, 108G/285L,108G/320W/323I, 108G/377I, 108G/377L, 126C,126C/184S/213S/280G/281P/285L/320G, 126C/184S/213S/372L,126C/189I/285L/372L, 126C/215P, 126C/372L, 181L/215P, 189E/372L/377Y,189I, 189I/214R/215P/271Y/282G, 189I/215K, 189I/215P/343V/372L,189I/215R/249T/277M, 189I/270E/285L, 189I/270E/372L, 189I/280G/282A,189I/320W/377I, 189I/343V, 189I/377I, 189L, 189Q, 189Q/214R,189Q/215P/271Y/281P/282C/377Y, 189Q/343V, 189Q/343V/377Y, 189Q/381L,213S/215P/320G, 214R/215P/271Y, 214R/215P/271Y/377Y, 214R/271Y,214R/280G/282A/343V/377Y/381L, 215K, 215K/281P/285L/372L,215K/281P/373G, 215K/285L/317P, 215K/285L/445L, 215K/323Y, 215K/372L,215K/372L/377L, 215K/373G, 215P, 215P/271Y/372L, 215P/320G,215P/320G/372L, 215P/372L, 215P/372L/377L, 215P/377L, 215P/381L, 215R,215R/249T/280G/281P/285L/372L, 215R/280G/281P/285L/372L,215R/281P/285L/373G, 215R/285P, 215R/320G, 215R/372L, 215W,215W/285L/346S, 215W/285P, 215W/373G, 249T/377L,269L/270E/281P/372L/377L, 270E/377L, 271Y, 271Y/343V, 271Y/343V/372L,271Y/343V/372L/381L, 280G/285L/372L, 281P/372L, 282A/285L/320W/323I,285L/323I, 320W, 343V/372L, 372L, 372L/377L, 372L/381L, 372M, 373G, and377L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 8. In some further embodiments, atleast one substitution or substitution set in the polypeptide sequencecomprises substitutions selected from P63A, P63A/M189A,P63A/N215R/A343V, P63R, P63R/Y65G/A108G, P63R/Y65G/A108G/M189L,P63R/Y65G/A108G/F377I, P63R/Y65G/T282A/A285L/Y320W/F323I,P63R/Y65G/Y320W/F323I, P63R/A108G, P63R/A108G/T282A/A285L/F377L,P63R/A108G/A285L/F377I, P63R/A108G/Y320W/F323C, P63R/F377I, P63T/N215R,P63Y, P63Y/M189L, P63Y/G212P/N215R, P63Y/G212P/N215R/P268A/A269N/A343V,P63Y/N215P/A269N, P63Y/N215R, P63Y/N215R/V270I/W271S,P63Y/P268A/A269N/V270I/A429V, Y65G/Y320W, Y65G/Y320W/F323I,Y65W/I69L/I372L, Y65W/I69M/G70A/L281P/I372L, Y65W/I69W/G70L/I372L,Y65W/G70L/I372M, A68P, A68P/I69L/M189E/G214R/W271Y/V280G,A68P/I69L/M189E/G214R/I372L, A68P/I69L/M189I/G214R/N215P/W271Y,A68P/I69L/M189I/L281P/T282C/I372L/F377Y/A381L, A68P/I69L/M189Q/G214R,A68P/I69L/M189Q/W271Y/V280G/I372L/A381L, A68P/I69L/N215P,A68P/I69L/W271Y, A68P/I69L/T282C/V287I, A68P/I69L/A343V/I372L,A68P/I69W/M189E/G214R/N215P/W271Y/L281P/T282G/A343V/A381L,A68P/I69W/M189E/V280G/L281P/T282A/I372L/F377Y,A68P/I69W/M189E/A343V/A381L, A68P/I69W/M189I/G214R/N215P,A68P/I69W/M189I/G214R/F377Y/A381L, A68P/I69W/M189I/W271Y,A68P/I69W/M189I/I372L, A68P/I69W/M189I/A381L,A68P/I69W/G214R/N215P/W271Y, A68P/I69W/G214R/A343V, A68P/I69W/N215P,A68P/A108G/F377L, A68P/E184S, A68P/E184S/M189E, A68P/M189I/W271Y/I372L,A68P/M189I/A343V, A68P/G214R/N215P/W271Y/L281P/T282A/I372L,A68P/N215P/W271Y/A343V/I372L/A381L, A68P/N215P/F377L, A68P/W271Y/I372L,A68P/F377L, I69F/A108G/V270E/I372L/F377L, I69F/M189L, I69F/N215K,I69F/N215K/A269L/V270I/F377L, I69F/N215R, I69F/A285L/T373G, I69L,I69L/G70L/P331Q/I372M, I69L/M189E/W271Y/L281P/T282A, I69L/M189I,I69L/M189I/G214R/W271Y/L281P/T282A/A343V, I69L/M189I/W271Y/A343V/A381L,I69L/M189I/V280G/T282G/A343V/I372L/A381L, I69L/M189I/T282A,I69L/M189Q/F377Y, I69L/N215P/W271Y/V280G/L281P/T282C,I69L/N215P/W271Y/T282A, I69L/N215P/W271Y/I372L,I69L/N215P/A343V/I372L/A381L, I69L/N215R/A285P/T317P, I69L/W271Y,I69L/W271Y/I372L, I69L/T282C/A343V/I372L, I69L/I372L, I69M/G70A/I372M,I69W, I69W/G70L, I69W/G70L/I372M, I69W/G70L/G459R, I69W/A108S,I69W/M189E/G214R/A343V/I372L, I69W/M189E/W271Y/A343V, I69W/M189E/I372L,I69W/M189I, I69W/M189I/N215P/A343V, I69W/M189I/W271Y,I69W/M189I/A343V/A381L, I69W/M189Q/I372L/F377Y,I69W/G212A/A213L/N215R/V280G/L281P,I69W/G214R/N215P/W271Y/I372L/F377Y/A381L, I69W/G214R/W271Y/T282A,I69W/G214R/W271Y/A343V, I69W/N215K/A343V, I69W/N215P, I69W/N215R,I69W/N215R/F323Y, I69W/T282A, I69W/I372M, I69Y/A108G/L281P/A285P,I69Y/T110A/N215R/L281P, I69Y/M189L/L281P/T373G, G70L, G70L/G212P, A108G,A108G/M189I/T282A/A285L/Y320W, A108G/M189L, A108G/M189L/Y320W,A108G/M189L/F377I, A108G/N215K, A108G/N215P/F377L, A108G/A269L/V270E,A108G/V270E, A108G/T282A/A285L/F377L, A108G/A285L, A108G/Y320W/F323I,A108G/F377I, A108G/F377L, R126C,R126C/E184S/A213S/V280G/L281P/A285L/Y320G, R126C/E184S/A213S/I372L,R126C/M189I/A285L/I372L, R126C/N215P, R126C/I372L, V181L/N215P,M189E/I372L/F377Y, M189I, M189I/G214R/N215P/W271Y/T282G, M189I/N215K,M189I/N215P/A343V/I372L, M189I/N215R/A249T/G277M, M189I/V270E/A285L,M189I/V270E/I372L, M189I/V280G/T282A, M189I/Y320W/F377I, M189I/A343V,M189I/F377I, M189L, M189Q, M189Q/G214R,M189Q/N215P/W271Y/L281P/T282C/F377Y, M189Q/A343V, M189Q/A343V/F377Y,M189Q/A381L, A213S/N215P/Y320G, G214R/N215P/W271Y,G214R/N215P/W271Y/F377Y, G214R/W271Y,G214R/V280G/T282A/A343V/F377Y/A381L, N215K, N215K/L281P/A285L/I372L,N215K/L281P/T373G, N215K/A285L/T317P, N215K/A285L/V445L, N215K/F323Y,N215K/I372L, N215K/I372L/F377L, N215K/T373G, N215P, N215P/W271Y/I372L,N215P/Y320G, N215P/Y320G/I372L, N215P/I372L, N215P/I372L/F377L,N215P/F377L, N215P/A381L, N215R, N215R/A249T/V280G/L281P/A285L/I372L,N215R/V280G/L281P/A285L/I372L, N215R/L281P/A285L/T373G, N215R/A285P,N215R/Y320G, N215R/I372L, N215W, N215W/A285L/G346S, N215W/A285P,N215W/T373G, A249T/F377L, A269L/V270E/L281P/I372L/F377L, V270E/F377L,W271Y, W271Y/A343V, W271Y/A343V/I372L, W271Y/A343V/I372L/A381L,V280G/A285L/I372L, L281P/I372L, T282A/A285L/Y320W/F323I, A285L/F323I,Y320W, A343V/I372L, I372L, I372L/F377L, I372L/A381L, I372M, T373G, andF377L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 8.

In some embodiments, the present invention also provides engineeredcarboxyesterase polypeptides that comprise a fragment of any of theengineered carboxyesterase polypeptides described herein that retainsthe functional carboxyesterase activity and/or improved property of thatengineered carboxyesterase polypeptide. Accordingly, in someembodiments, the present invention provides a polypeptide fragmenthaving carboxyesterase activity (e.g., capable of converting substrateto product under suitable reaction conditions), wherein the fragmentcomprises at least about 80%, 90%, 95%, 98%, or 99% of a full-lengthamino acid sequence of an engineered polypeptide of the presentinvention, such as an exemplary engineered polypeptide of having theeven-numbered sequence identifiers of SEQ ID NOs: 2-136.

In some embodiments, the engineered carboxyesterase polypeptide of theinvention comprises an amino acid sequence comprising at least onedeletion, addition, and/or substitution, as compared to any one of theengineered carboxyesterase polypeptide sequences described herein, suchas the exemplary engineered polypeptide sequences having theeven-numbered sequence identifiers of SEQ ID NOs: 2-136. Thus, for eachand every embodiment of the engineered carboxyesterase polypeptides ofthe invention, the amino acid sequence can comprise deletions,additions, and/or substitutions of one or more amino acids, 2 or moreamino acids, 3 or more amino acids, 4 or more amino acids, 5 or moreamino acids, 6 or more amino acids, 8 or more amino acids, 10 or moreamino acids, 15 or more amino acids, or 20 or more amino acids, up to10% of the total number of amino acids, up to 10% of the total number ofamino acids, up to 20% of the total number of amino acids, or up to 30%of the total number of amino acids of the carboxyesterase polypeptides,where the associated functional activity and/or improved properties ofthe engineered carboxyesterase described herein is maintained. In someembodiments, the deletions, additions, and/or substitutions can comprise1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22,1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 additions,and/or substitutions of the amino acid residues. In some embodiments,the number of deletions, additions, and/or substitutions can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 30, 35, 40, 45, 50, 55, or 60 of the amino acidresidues. In some embodiments, the deletions, additions, and/orsubstitutions can comprise deletions, additions, and/or substitutions of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22,23, 24, 25 or 30 amino acid residues.

In some embodiments, the present invention provides an engineeredcarboxyesterase polypeptide having an amino acid sequence comprising aninsertion as compared to any one of the engineered carboxyesterasepolypeptide sequences described herein, such as the exemplary engineeredpolypeptide sequences having the even-numbered sequence identifiers ofSEQ ID NO: 2-136. Thus, for each and every embodiment of thecarboxyesterase polypeptides of the invention, the insertions cancomprise one or more amino acids, 2 or more amino acids, 3 or more aminoacids, 4 or more amino acids, 5 or more amino acids, 6 or more aminoacids, 8 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, where the associated functionalactivity and/or improved properties of the engineered carboxyesterasedescribed herein is maintained. The insertions can be to amino orcarboxy terminus, or internal portions of the carboxyesterasepolypeptide.

In some embodiments, the polypeptides of the present invention are inthe form of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purification sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

The engineered carboxyesterase polypeptides described herein are notrestricted to the genetically encoded amino acids. Thus, in addition tothe genetically encoded amino acids, the polypeptides described hereinmay be comprised, either in whole or in part, of naturally-occurringand/or synthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereoisomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5f0; styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art. These amino acids may be in either the L- orD-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

As will be apparent to the skilled artisan, the foregoing residuepositions and the specific amino acid residues for each residue positioncan be used individually or in various combinations to synthesizecarboxyesterase polypeptides having desired improved properties,including, among others, enzyme activity, substrate/product preference,stereoselectivity, substrate/product tolerance, and stability undervarious conditions, such as increased temperature, solvent, and/or pH.

The engineered carboxyesterase polypeptides of the present inventionwere generated by directed evolution of SEQ ID NO: 2 for efficientamidation of substrates of interest to products of interest, undercertain industrially relevant conditions and have one or more residuedifferences as compared to a reference carboxyesterase polypeptide.These residue differences are associated with improvements in variousenzyme properties, particularly increased activity, increased solventtolerance, and reduced toxicity to host cells (e.g., E. coli). In someadditional embodiments, the variant carboxyesterases also exhibitedincreased stereoselectivity, increased stability, and tolerance ofincreased substrate and/or product concentration (e.g., decreasedproduct inhibition). Accordingly, in some embodiments, the engineeredpolypeptides having carboxyesterase activity are capable of convertingthe substrate compound(s) to product(s) with an activity that isincreased at least about 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold,500 fold, 1000 fold, or more relative to the activity of wild-type T.fusca carboxyesterase polypeptide (e.g., SEQ ID NO: 2), under suitablereaction conditions. In some embodiments, the engineered polypeptideshaving carboxyesterase activity are capable of converting substrate toproduct with a percent conversion of at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, in areaction time of about 48 h, about 36 h, about 24 h, or even a shorterlength of time, under suitable reaction conditions. In some embodiments,the engineered polypeptides having carboxyesterase activity are capableof converting substrate to product diastereomeric excess of at least90%, 95%, 97%, 98%, 99%, or greater, under suitable reaction conditions.

In some embodiments, the engineered polypeptides having carboxyesteraseactivity are capable of converting substrate to product with increasedtolerance for the presence of the substrate relative to the substratetolerance of a reference polypeptide (e.g., SEQ ID NO: 2), undersuitable reaction conditions. Accordingly, in some embodiments theengineered polypeptides are capable of converting the substrate ofsubstrate to product in the presence of a substrate loadingconcentration of at least about 1 g/L, 5 g/L, 10 g/L, 20 g/L, about 30g/L, about 40 g/L, about 50 g/L, about 70 g/L, about 75 g/L, about 100g/L, with a percent conversion of at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 91%, at least about 92%, at least about94%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%, in areaction time of about 72h, about 48h, about 36h, about 24 h, or evenshorter length of time, under suitable reaction conditions.

Some suitable reaction conditions under which the above-describedimproved properties of the engineered polypeptides can be determinedwith respect to concentrations or amounts of polypeptide, substrate,buffer, co-solvent, pH, and/or conditions including temperature andreaction time are provided herein. In some embodiments, the suitablereaction conditions comprise the assay conditions described below and inthe Examples.

In some embodiments, the engineered polypeptides can be provided on asolid support, such as a membrane, resin, solid carrier, or other solidphase material. A solid support can be composed of organic polymers suchas polystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of a solid support can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation.

In some embodiments, the engineered polypeptides having carboxyesteraseactivity are bound or immobilized on the solid support such that theyretain at least a portion of their improved properties relative to areference polypeptide (e.g., SEQ ID NO: 2). In such embodiments, theimmobilized polypeptides can facilitate the biocatalytic conversion ofthe substrate compound to the desired product, and after the reaction iscomplete are easily retained (e.g., by retaining beads on whichpolypeptide is immobilized) and then reused or recycled in subsequentreactions. Such immobilized enzyme processes allow for furtherefficiency and cost reduction. Accordingly, it is further contemplatedthat any of the methods of using the engineered carboxyesterasepolypeptides of the present invention can be carried out using the samecarboxyesterase polypeptides bound or immobilized on a solid support.

The engineered carboxyesterase polypeptide can be bound non-covalentlyor covalently. Various methods for conjugation and immobilization ofenzymes to solid supports (e.g., resins, membranes, beads, glass, etc.)are well known in the art. Other methods for conjugation andimmobilization of enzymes to solid supports (e.g., resins, membranes,beads, glass, etc.) are well known in the art (See, e.g., Yi et al.,Proc. Biochem., 42: 895-898 [2007]; Martin et al., Appl. Microbiol.Biotechnol., 76: 843-851 [2007]; Koszelewski et al., J. Mol. Cat. B:Enz., 63: 39-44 [2010]; Truppo et al., Org. Proc. Res. Develop.,published online: dx.doi.org/10.1021/op200157c; and Mateo et al.,Biotechnol. Prog., 18:629-34 [2002], etc.). Solid supports useful forimmobilizing the engineered carboxyesterase polypeptides of the presentinvention include, but are not limited to, beads or resins comprisingpolymethacrylate with epoxide functional groups, polymethacrylate withamino epoxide functional groups, styrene/DVB copolymer orpolymethacrylate with octadecyl functional groups. Exemplary solidsupports useful for immobilizing the engineered carboxyesterases of thepresent invention include, but are not limited to, chitosan beads,Eupergit C, and SEPABEADs (Mitsubishi), including the followingdifferent types of SEPABEAD: EC-EP, EC-HFA/S, EXA252, EXE119 and EXE120.

In some embodiments, the engineered carboxyesterase polypeptides can beprovided in the form of an array in which the polypeptides are arrangedin positionally distinct locations. In some embodiments, thepositionally distinct locations are wells in a solid support such as a96-well plate. A plurality of supports can be configured on an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments. Such arrays can be used to test avariety of substrate compounds for conversion by the polypeptides.

In some embodiments, the engineered polypeptides described herein can beprovided in the form of kits. The polypeptides in the kits may bepresent individually or as a plurality of polypeptides. The kits canfurther include reagents for carrying out enzymatic reactions,substrates for assessing the activity of polypeptides, as well asreagents for detecting the products. The kits can also include reagentdispensers and instructions for use of the kits. In some embodiments,the kits of the present invention include arrays comprising a pluralityof different engineered carboxyesterase polypeptides at differentaddressable position, wherein the different polypeptides are differentvariants of a reference sequence each having at least one differentimproved enzyme property. Such arrays comprising a plurality ofengineered polypeptides and methods of their use are known (See, e.g.,WO2009/008908A2).

Polynucleotides Encoding Engineered Carboxyesterases

In another aspect, the present invention provides polynucleotidesencoding the engineered carboxyesterase enzymes. The polynucleotides maybe operatively linked to one or more heterologous regulatory sequencesthat control gene expression to create a recombinant polynucleotidecapable of expressing the polypeptide. Expression constructs containinga heterologous polynucleotide encoding the engineered carboxyesterasecan be introduced into appropriate host cells to express thecorresponding carboxyesterase polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improvedcarboxyesterase enzymes disclosed herein. Thus, having identified aparticular amino acid sequence, those skilled in the art could make anynumber of different nucleic acids by simply modifying the sequence ofone or more codons in a way which does not change the amino acidsequence of the protein. In this regard, the present inventionspecifically contemplates each and every possible variation ofpolynucleotides that could be made by selecting combinations based onthe possible codon choices, and all such variations are to be consideredspecifically disclosed for any polypeptide disclosed herein, includingthe amino acid sequences presented in the Tables in the Examples. Invarious embodiments, the codons are preferably selected to fit the hostcell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. By way of example, the polynucleotide of SEQ ID NO: 1 has beencodon optimized for expression in E. coli, but otherwise encodes thenaturally occurring carboxyesterase of T. fusca.

In some embodiments, the polynucleotide encodes an engineeredcarboxyesterase polypeptide comprising an amino acid sequence selectedfrom SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, or 136.

In some embodiments, the polynucleotides encoding the engineeredcarboxyesterases or a functional fragment thereof, are selected frompolynucleotide sequences comprising at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, and/or 139.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide comprising SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, and/or 139.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% or more sequence identity at the nucleotide level to a referencepolynucleotide encoding the engineered carboxyesterase. In someembodiments, the reference polynucleotide comprises SEQ ID NO: 1, whilein some other embodiments, the reference polynucloeotide comprises SEQID NO:137. In some further embodiments, the reference polynucleotidesequence comprises SEQ ID NO:139. In some additional embodiments, theengineered carboxyesterase sequences comprise sequences that comprisepositions identified to be beneficial, as described in the Examples.

The present invention also provides polynucleotide sequences encoding atleast one engineered carboxyesterase provided herein. In someembodiments, the polynucleotide sequences encode at least one engineeredcarboxyesterase comprising a polypeptide sequence having at least 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 2 or a functional fragmentthereof, wherein the engineered carboxyesterase comprises at least onesubstitution or substitution set in its polypeptide sequence, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2. In some embodiments, the polynucleotidesequences encode at least one engineered carboxyesterase comprisingsubstitutions at positions selected from: 39, 39/323, 62, 62/117, 63,64, 65, 66, 68, 69, 70, 71, 71/263, 77, 77/184, 103, 103/147, 104,104/429, 105, 107, 107/185, 108, 109, 109/117, 110, 111, 113, 114, 115,117, 118, 118/269, 118/349, 119, 126, 147, 153, 153/215, 164, 164/271,174, 174/282, 183, 184, 184/249, 185, 186, 187, 188, 190, 209, 210, 211,212, 213, 213/271, 213/345, 214, 215, 215/271, 216, 217, 217/231, 224,224/268/372, 231, 249, 249/284, 263, 268, 269, 270, 270/470, 271,271/416, 276, 277, 278, 279, 279/280/282, 280, 281, 281/374, 282, 283,283/429, 284, 284/438, 285, 286, 311, 317, 320, 320/323, 320/323/372,320/372/376, 320/376/377, 321, 323, 324, 345, 349, 372, 372/376, 373,374, 376, 377, 405, 416, 420, 427, 428, 429, 438, and 470, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 2. In some additional embodiments, thepolynucleotide sequences encode at least one engineered carboxyesterasecomprising at least one substitution or substitution set selected from:39/323, 62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263, 77/184, 103,103/147, 104, 104/429, 105, 107, 107/185, 108, 109/117, 110, 111, 113,114, 115, 117, 118, 118/269, 118/349, 119, 126, 153, 153/215, 164/271,174/282, 183, 184, 184/249, 185, 186, 187, 188, 190, 209, 210, 211, 212,213, 213/271, 213/345, 214, 215, 215/271, 216, 217, 217/231,224/268/372, 249/284, 269, 270, 270/470, 271, 271/416, 276, 277, 278,279, 279/280/282, 280, 281, 281/374, 282, 283, 283/429, 284, 284/438,285, 286, 311, 317, 320, 320/323, 320/323/372, 320/372/376, 320/376/377,321, 323, 324, 372, 372/376, 373, 376, 377, 405, 420, 427, 428, and 429,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 2. In some additional embodiments,the polynucleotide sequences encode at least one engineeredcarboxyesterase comprising at least one substitution or substitution setselected from: 39M/323I, 62H/117G, 63A, 63R, 63T, 63Y, 64A, 64E, 64G,64I, 64T, 64V, 64W, 65G, 65S, 65T, 65W, 66N, 68L, 68P, 69F, 69G, 69H,69L, 69V, 69W, 69Y, 70L, 70R, 70T, 70W, 71F, 71G, 71H/263R, 71P, 71R,71V, 71Y, 77S/184G, 103P, 103R, 103T/147S, 104P, 104Q/429V, 105L,107D/185W, 107L, 107P, 107S, 108G, 108K, 108Q, 108R, 108S, 108W,109G/117M, 110A, 110H, 110P, 110S, 111L, 111M, 111R, 111S, 111V, 111W,113P, 114A, 114H, 114Q, 115H, 115T, 115V, 117A, 117F, 118G/349V, 118I,118N, 118N/269T, 119G, 119P, 119S, 126C, 153H/215P, 153L, 164R/271T,174D/282V, 183P, 184F, 184G, 184P, 184S/249T, 184Y, 185A, 185T, 186C,186G, 186P, 186R, 186T, 187P, 188E, 188G, 190H, 190K, 190L, 190M, 190Q,190R, 190W, 209E, 209G, 209P, 209S, 209V, 210P, 210T, 210W, 211I, 211L,211R, 211V, 212A, 212P, 212R, 212S, 213C, 213E, 213L, 213N, 213P, 213Q,213R/345G, 213S, 213T/271K, 213V, 214K, 214L, 214T, 214V, 215K, 215M,215P, 215R, 215R/271R, 215W, 216P, 217G, 217L, 217P, 217R, 217R/231V,217S, 217V, 217W, 224I/268S/372F, 249V/284P, 269N, 269V, 270I,270I/470M, 270R, 271A, 271K, 271L, 271P, 271Q/416V, 271S, 271T, 276F,277M, 278H, 278S, 279C, 279E, 279G, 279L/280G/282M, 279V, 280E, 280G,280S, 281P, 281V, 281Y/374N, 282A, 282C, 282Q, 282R, 282S, 282T, 282W,283C, 283D, 283K, 283R/429V, 283T, 283V, 283Y, 284C, 284T, 284T/438T,284V, 285L, 285M, 285P, 286V, 311I, 317C, 317P, 320A, 320F, 320G,320G/323S, 320S, 320S/323S/372A, 320S/372A/376G, 320S/376G/377V, 320W,321L, 321S, 323C, 323I, 323R, 323Y, 324A, 372A/376A, 372L, 373G, 376A,376G, 376L, 376M, 377L, 377W, 377Y, 405D, 420G, 427A, 428V, and 429L,wherein the amino acid positions are numbered with reference to SEQ IDNO: 2. In some further embodiments, the polynucleotide sequences encodeat least one engineered carboxyesterase comprising at least onesubstitution or substitution set selected from: T39M/F323I, R62H/P117G,P63A, P63R, P63T, P63Y, P64A, P64E, P64G, P64I, P64T, P64V, P64W, Y65G,Y65S, Y65T, Y65W, P66N, A68L, A68P, I69F, I69G, I69H, I69L, I69V, I69W,I69Y, G70L, G70R, G70T, G70W, A71F, A71G, A71H/Q263R, A71P, A71R, A71V,A71Y, F77S/E184G, W103P, W103R, W103T/P147S, I104P, I104Q/A429V, H105L,G107D/S185W, G107L, G107P, G107S, A108G, A108K, A108Q, A108R, A108S,A108W, F109G/P117M, T110A, T110H, T110P, T110S, N111L, N111M, N111R,N111S, N111V, N111W, S113P, G114A, G114H, G114Q, S115H, S115T, S115V,P117A, P117F, V118G/A349V, V118I, V118N, V118N/A269T, Y119G, Y119P,Y119S, R126C, R153H/N215P, R153L, W164R/W271T, G174D/L282V, G183P,E184F, E184G, E184P, E184S/A249T, E184Y, S185A, S185T, A186C, A186G,A186P, A186R, A186T, G187P, A188E, A188G, S190H, S190K, S190L, S190M,S190Q, S190R, S190W, L209E, L209G, L209P, L209S, L209V, Q210P, Q210T,Q210W, S211I, S211L, S211R, S211V, G212A, G212P, G212R, G212S, A213C,A213E, A213L, A213N, A213P, A213Q, A213R/S345G, A213S, A213T/W271K,A213V, G214K, G214L, G214T, G214V, N215K, N215M, N215P, N215R,N215R/W271R, N215W, M216P, A217G, A217L, A217P, A217R, A217R/A231V,A217S, A217V, A217W, T224I/P268S/I372F, A249V/F284P, A269N, A269V,V270I, V270I/V470M, V270R, W271A, W271K, W271L, W271P, W271Q/A416V,W271S, W271T, A276F, G277M, G278H, G278S, S279C, S279E, S279G,S279L/V280G/L282M, S279V, V280E, V280G, V280S, L281P, L281V,L281Y/D374N, L282A, L282C, L282Q, L282R, L282S, L282T, L282W, P283C,P283D, P283K, P283R/A429V, P283T, P283V, P283Y, F284C, F284T,F284T/P438T, F284V, A285L, A285M, A285P, P286V, L311I, T317C, T317P,Y320A, Y320F, Y320G, Y320G/F323S, Y320S, Y320S/F323S/I372A,Y320S/I372A/V376G, Y320S/V376G/F377V, Y320W, R321L, R321S, F323C, F323I,F323R, F323Y, L324A, I372A/V376A, I372L, T373G, V376A, V376G, V376L,V376M, F377L, F377W, F377Y, P405D, P420G, D427A, R428V, and A429L,wherein the amino acids are numbered with reference to SEQ ID NO: 2. Insome embodiments, the polynucleotide sequences encode at least oneengineered carboxyesterase comprising a substitution at position 282,wherein the position is numbered with reference to SEQ ID NO: 2. In somefurther embodiments, the substitution at position 282 is aliphatic,non-polar, basic, polar, or aromatic. In yet some additionalembodiments, the substitution is selected from X282T, X282G, X282A,X282V, X282M, X282C, X282W, X282Q, X282S, X282T, and X282R.

The present invention also provides polynucleotide sequences encoding atleast one engineered carboxyesterase comprising a polypeptide sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8 or afunctional fragment thereof, wherein the engineered carboxyesterasecomprises at least one substitution or substitution set in itspolypeptide sequence, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 8. Insome embodiments, the polynucleotide sequences encode engineeredcarboxyesterases comprising at least one substitution or substitutionset at positions selected from: 63, 63/65/108, 63/65/108/189,63/65/108/377, 63/65/282/285/320/323, 63/65/320/323, 63/108,63/108/282/285/377, 63/108/285/377, 63/108/320/323, 63/189, 63/212/215,63/212/215/268/269/343, 63/215, 63/215/269, 63/215/270/271, 63/215/343,63/268/269/270/429, 63/377, 65/69/70/281/372, 65/69/70/372, 65/69/372,65/70/372, 65/320, 65/320/323, 68, 68/69/189/214, 68/69/189/214/215,68/69/189/214/215/271, 68/69/189/214/215/271/281/282/343/381,68/69/189/214/271/280, 68/69/189/214/372, 68/69/189/214/377/381,68/69/189/271, 68/69/189/271/280/372/381, 68/69/189/280/281/282/372/377,68/69/189/281/282/372/377/381, 68/69/189/343/381, 68/69/189/372,68/69/189/381, 68/69/214/215/271, 68/69/214/343, 68/69/215, 68/69/271,68/69/282/287, 68/69/343/372, 68/108/377, 68/184, 68/184/189,68/189/271/372, 68/189/343, 68/214/215/271/281/282/372,68/215/271/343/372/381, 68/215/377, 68/271/372, 68/377, 69, 69/70,69/70/331/372, 69/70/372, 69/70/459, 69/108, 69/108/270/372/377,69/108/281/285, 69/110/215/281, 69/189, 69/189/214/271/281/282/343,69/189/214/343/372, 69/189/215/343, 69/189/271, 69/189/271/281/282,69/189/271/343, 69/189/271/343/381, 69/189/280/282/343/372/381,69/189/281/373, 69/189/282, 69/189/343/381, 69/189/372, 69/189/372/377,69/189/377, 69/212/213/215/280/281, 69/214/215/271/372/377/381,69/214/271/282, 69/214/271/343, 69/215, 69/215/269/270/377,69/215/271/280/281/282, 69/215/271/282, 69/215/271/372, 69/215/285/317,69/215/323, 69/215/343, 69/215/343/372/381, 69/271, 69/271/372, 69/282,69/282/343/372, 69/285/373, 69/372, 70, 70/212, 108, 108/189,108/189/282/285/320, 108/189/320, 108/189/377, 108/215, 108/215/377,108/269/270, 108/270, 108/282/285/377, 108/285, 108/320/323, 108/377,126, 126/184/213/280/281/285/320, 126/184/213/372, 126/189/285/372,126/215, 126/372, 181/215, 189, 189/214, 189/214/215/271/282, 189/215,189/215/249/277, 189/215/271/281/282/377, 189/215/343/372, 189/270/285,189/270/372, 189/280/282, 189/320/377, 189/343, 189/343/377,189/372/377, 189/377, 189/381, 213/215/320, 214/215/271,214/215/271/377, 214/271, 214/280/282/343/377/381, 215,215/249/280/281/285/372, 215/271/372, 215/280/281/285/372,215/281/285/372, 215/281/285/373, 215/281/373, 215/285, 215/285/317,215/285/346, 215/285/445, 215/320, 215/320/372, 215/323, 215/372,215/372/377, 215/373, 215/377, 215/381, 249/377, 269/270/281/372/377,270/377, 271, 271/343, 271/343/372, 271/343/372/381, 280/285/372,281/372, 282/285/320/323, 285/323, 320, 343/372, 372, 372/377, 372/381,373, and 377, wherein the amino acid positions are numbered withreference to SEQ ID NO: 8. In some further embodiments, thepolynucleotide sequence encodes an engineered carboxyesterase comprisingat least one substitution or substitution set selected from: 63A,63A/189A, 63A/215R/343V, 63R, 63R/65G/108G, 63R/65G/108G/189L,63R/65G/108G/377I, 63R/65G/282A/285L/320W/323I, 63R/65G/320W/323I,63R/108G, 63R/108G/282A/285L/377L, 63R/108G/285L/377I,63R/108G/320W/323C, 63R/377I, 63T/215R, 63Y, 63Y/189L, 63Y/212P/215R,63Y/212P/215R/268A/269N/343V, 63Y/215P/269N, 63Y/215R,63Y/215R/270I/271S, 63Y/268A/269N/270I/429V, 65G/320W, 65G/320W/323I,65W/69L/372L, 65W/69M/70A/281P/372L, 65W/69W/70L/372L, 65W/70L/372M,68P, 68P/69L/189E/214R/271Y/280G, 68P/69L/189E/214R/372L,68P/69L/189I/214R/215P/271Y, 68P/69L/189I/281P/282C/372L/377Y/381L,68P/69L/189Q/214R, 68P/69L/189Q/271Y/280G/372L/381L, 68P/69L/215P,68P/69L/271Y, 68P/69L/282C/287I, 68P/69L/343V/372L,68P/69W/189E/214R/215P/271Y/281P/282G/343V/381L,68P/69W/189E/280G/281P/282A/372L/377Y, 68P/69W/189E/343V/381L,68P/69W/189I/214R/215P, 68P/69W/189I/214R/377Y/381L, 68P/69W/189I/271Y,68P/69W/189I/372L, 68P/69W/189I/381L, 68P/69W/214R/215P/271Y,68P/69W/214R/343V, 68P/69W/215P, 68P/108G/377L, 68P/184S, 68P/184S/189E,68P/189I/271Y/372L, 68P/189I/343V, 68P/214R/215P/271Y/281P/282A/372L,68P/215P/271Y/343V/372L/381L, 68P/215P/377L, 68P/271Y/372L, 68P/377L,69F/108G/270E/372L/377L, 69F/189L, 69F/215K, 69F/215K/269L/270I/377L,69F/215R, 69F/285L/373G, 69L, 69L/70L/331Q/372M,69L/189E/271Y/281P/282A, 69L/189I, 69L/189I/214R/271Y/281P/282A/343V,69L/189I/271Y/343V/381L, 69L/189I/280G/282G/343V/372L/381L,69L/189I/282A, 69L/189Q/377Y, 69L/215P/271Y/280G/281P/282C,69L/215P/271Y/282A, 69L/215P/271Y/372L, 69L/215P/343V/372L/381L,69L/215R/285P/317P, 69L/271Y, 69L/271Y/372L, 69L/282C/343V/372L,69L/372L, 69M/70A/372M, 69W, 69W/70L, 69W/70L/372M, 69W/70L/459R,69W/108S, 69W/189E/214R/343V/372L, 69W/189E/271Y/343V, 69W/189E/372L,69W/189I, 69W/189I/215P/343V, 69W/189I/271Y, 69W/189I/343V/381L,69W/189Q/372L/377Y, 69W/212A/213L/215R/280G/281P,69W/214R/215P/271Y/372L/377Y/381L, 69W/214R/271Y/282A,69W/214R/271Y/343V, 69W/215K/343V, 69W/215P, 69W/215R, 69W/215R/323Y,69W/282A, 69W/372M, 69Y/108G/281P/285P, 69Y/110A/215R/281P,69Y/189L/281P/373G, 70L, 70L/212P, 108G, 108G/189I/282A/285L/320W,108G/189L, 108G/189L/320W, 108G/189L/377I, 108G/215K, 108G/215P/377L,108G/269L/270E, 108G/270E, 108G/282A/285L/377L, 108G/285L,108G/320W/323I, 108G/377I, 108G/377L, 126C,126C/184S/213S/280G/281P/285L/320G, 126C/184S/213S/372L,126C/189I/285L/372L, 126C/215P, 126C/372L, 181L/215P, 189E/372L/377Y,189I, 189I/214R/215P/271Y/282G, 189I/215K, 189I/215P/343V/372L,189I/215R/249T/277M, 189I/270E/285L, 189I/270E/372L, 189I/280G/282A,189I/320W/377I, 189I/343V, 189I/377I, 189L, 189Q, 189Q/214R,189Q/215P/271Y/281P/282C/377Y, 189Q/343V, 189Q/343V/377Y, 189Q/381L,213S/215P/320G, 214R/215P/271Y, 214R/215P/271Y/377Y, 214R/271Y,214R/280G/282A/343V/377Y/381L, 215K, 215K/281P/285L/372L,215K/281P/373G, 215K/285L/317P, 215K/285L/445L, 215K/323Y, 215K/372L,215K/372L/377L, 215K/373G, 215P, 215P/271Y/372L, 215P/320G,215P/320G/372L, 215P/372L, 215P/372L/377L, 215P/377L, 215P/381L, 215R,215R/249T/280G/281P/285L/372L, 215R/280G/281P/285L/372L,215R/281P/285L/373G, 215R/285P, 215R/320G, 215R/372L, 215W,215W/285L/346S, 215W/285P, 215W/373G, 249T/377L,269L/270E/281P/372L/377L, 270E/377L, 271Y, 271Y/343V, 271Y/343V/372L,271Y/343V/372L/381L, 280G/285L/372L, 281P/372L, 282A/285L/320W/323I,285L/323I, 320W, 343V/372L, 372L, 372L/377L, 372L/381L, 372M, 373G, and377L, wherein the amino acid positions are numbered with reference toSEQ ID NO: 8. In some further embodiments, the polynucleotide sequenceencodes an engineered carboxyesterase comprising at least onesubstitution or substitution set selected from P63A, P63A/M189A,P63A/N215R/A343V, P63R, P63R/Y65G/A108G, P63R/Y65G/A108G/M189L,P63R/Y65G/A108G/F377I, P63R/Y65G/T282A/A285L/Y320W/F323I,P63R/Y65G/Y320W/F323I, P63R/A108G, P63R/A108G/T282A/A285L/F377L,P63R/A108G/A285L/F377I, P63R/A108G/Y320W/F323C, P63R/F377I, P63T/N215R,P63Y, P63Y/M189L, P63Y/G212P/N215R, P63Y/G212P/N215R/P268A/A269N/A343V,P63Y/N215P/A269N, P63Y/N215R, P63Y/N215R/V270I/W271S,P63Y/P268A/A269N/V270I/A429V, Y65G/Y320W, Y65G/Y320W/F323I,Y65W/I69L/I372L, Y65W/I69M/G70A/L281P/I372L, Y65W/I69W/G70L/I372L,Y65W/G70L/I372M, A68P, A68P/I69L/M189E/G214R/W271Y/V280G,A68P/I69L/M189E/G214R/I372L, A68P/I69L/M189I/G214R/N215P/W271Y,A68P/I69L/M189I/L281P/T282C/I372L/F377Y/A381L, A68P/I69L/M189Q/G214R,A68P/I69L/M189Q/W271Y/V280G/I372L/A381L, A68P/I69L/N215P,A68P/I69L/W271Y, A68P/I69L/T282C/V287I, A68P/I69L/A343V/I372L,A68P/I69W/M189E/G214R/N215P/W271Y/L281P/T282G/A343V/A381L,A68P/I69W/M189E/V280G/L281P/T282A/I372L/F377Y,A68P/I69W/M189E/A343V/A381L, A68P/I69W/M189I/G214R/N215P,A68P/I69W/M189I/G214R/F377Y/A381L, A68P/I69W/M189I/W271Y,A68P/I69W/M189I/I372L, A68P/I69W/M189I/381L,A68P/I69W/G214R/N215P/W271Y, A68P/I69W/G214R/A343V, A68P/I69W/N215P,A68P/A108G/F377L, A68P/E184S, A68P/E184S/M189E, A68P/M189I/W271Y/I372L,A68P/M189I/A343V, A68P/G214R/N215P/W271Y/L281P/T282A/I372L,A68P/N215P/W271Y/A343V/I372L/A381L, A68P/N215P/F377L, A68P/W271Y/I372L,A68P/F377L, I69F/A108G/V270E/I372L/F377L, I69F/M189L, I69F/N215K,I69F/N215K/A269L/V270I/F377L, I69F/N215R, I69F/A285L/T373G, I69L,I69L/G70L/P331Q/I372M, I69L/M189E/W271Y/L281P/T282A, I69L/M189I,I69L/M189I/G214R/W271Y/L281P/T282A/A343V, I69L/M189I/W271Y/A343V/A381L,I69L/M189I/V280G/T282G/A343V/I372L/A381L, I69L/M189I/T282A,I69L/M189Q/F377Y, I69L/N215P/W271Y/V280G/L281P/T282C,I69L/N215P/W271Y/T282A, I69L/N215P/W271Y/I372L,I69L/N215P/A343V/I372L/A381L, I69L/N215R/A285P/T317P, I69L/W271Y,I69L/W271Y/I372L, I69L/T282C/A343V/I372L, I69L/I372L, I69M/G70A/I372M,I69W, I69W/G70L, I69W/G70L/I372M, I69W/G70L/G459R, I69W/A108S,I69W/M189E/G214R/A343V/I372L, I69W/M189E/W271Y/A343V, I69W/M189E/I372L,I69W/M189I, I69W/M189I/N215P/A343V, I69W/M189I/W271Y,I69W/M189I/A343V/A381L, I69W/M189Q/I372L/F377Y,I69W/G212A/A213L/N215R/V280G/L281P,I69W/G214R/N215P/W271Y/I372L/F377Y/A381L, I69W/G214R/W271Y/T282A,I69W/G214R/W271Y/A343V, I69W/N215K/A343V, I69W/N215P, I69W/N215R,I69W/N215R/F323Y, I69W/T282A, I69W/I372M, I69Y/A108G/L281P/A285P,I69Y/T110A/N215R/L281P, I69Y/M189L/L281P/T373G, G70L, G70L/G212P, A108G,A108G/M189I/T282A/A285L/Y320W, A108G/M189L, A108G/M189L/Y320W,A108G/M189L/F377I, A108G/N215K, A108G/N215P/F377L, A108G/A269L/V270E,A108G/V270E, A108G/T282A/A285L/F377L, A108G/A285L, A108G/Y320W/F323I,A108G/F377I, A108G/F377L, R126C,R126C/E184S/A213S/V280G/L281P/A285L/Y320G, R126C/E184S/A213S/I372L,R126C/M189I/A285L/I372L, R126C/N215P, R126C/I372L, V181L/N215P,M189E/I372L/F377Y, M189I, M189I/G214R/N215P/W271Y/T282G, M189I/N215K,M189I/N215P/A343V/I372L, M189I/N215R/A249T/G277M, M189I/V270E/A285L,M189I/V270E/I372L, M189I/V280G/T282A, M189I/Y320W/F377I, M189I/A343V,M189I/F377I, M189L, M189Q, M189Q/G214R,M189Q/N215P/W271Y/L281P/T282C/F377Y, M189Q/A343V, M189Q/A343V/F377Y,M189Q/A381L, A213S/N215P/Y320G, G214R/N215P/W271Y,G214R/N215P/W271Y/F377Y, G214R/W271Y,G214R/V280G/T282A/A343V/F377Y/A381L, N215K, N215K/L281P/A285L/I372L,N215K/L281P/T373G, N215K/A285L/T317P, N215K/A285L/V445L, N215K/F323Y,N215K/I372L, N215K/I372L/F377L, N215K/T373G, N215P, N215P/W271Y/I372L,N215P/Y320G, N215P/Y320G/I372L, N215P/I372L, N215P/I372L/F377L,N215P/F377L, N215P/A381L, N215R, N215R/A249T/V280G/L281P/A285L/I372L,N215R/V280G/L281P/A285L/I372L, N215R/L281P/A285L/T373G, N215R/A285P,N215R/Y320G, N215R/I372L, N215W, N215W/A285L/G346S, N215W/A285P,N215W/T373G, A249T/F377L, A269L/V270E/L281P/I372L/F377L, V270E/F377L,W271Y, W271Y/A343V, W271Y/A343V/I372L, W271Y/A343V/I372L/A381L,V280G/A285L/I372L, L281P/I372L, T282A/A285L/Y320W/F323I, A285L/F323I,Y320W, A343V/I372L, I372L, I372L/F377L, I372L/A381L, I372M, T373G, andF377L, wherein the amino acid positions are numbered with reference toSEQ ID NO: 8.

An isolated polynucleotide encoding an improved carboxyesterasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art.

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present invention, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (See, e.g., Villa-Kamaroff et al., Proc.Natl. Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter(See, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25 [1983]).Additional suitable promoters are known to those in the art.

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present inventioninclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters include, but are not limited to thosefrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase, aswell as other useful promoters for yeast host cells (See, e.g., Romanos,et al., Yeast 8:423-488 [1992]).

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase, as well as other usefulterminators for yeast host cells known in the art (See, e.g., Romanos etal., supra).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase., as well asadditional useful polyadenylation sequences for yeast host cells knownin the art (See, e.g., Guo et al., Mol. Cell. Biol., 15:5983-5990[1995]).

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NC1B11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA—as well as additional signalpeptides known in the art (See, e.g., Simonen et al., Microbiol. Rev.,57: 109-137 [1993]).

Effective signal peptide coding regions for filamentous fungal hostcells include, but are not limited to the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa lipase. Useful signal peptides for yeast host cells can befrom the genes for Saccharomyces cerevisiae alpha-factor andSaccharomyces cerevisiae invertase, as well as additional useful signalpeptide coding regions (See, e.g., Romanos et al., 1992, supra).

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the carboxyesterasepolypeptide of the present invention would be operably linked with theregulatory sequence.

Thus, in some embodiments, the present invention is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered carboxyesterase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The various nucleic acid and controlsequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present invention may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector (i.e., avector that exists as an extrachromosomal entity), the replication ofwhich is independent of chromosomal replication, (e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificialchromosome). The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker can be a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like. Examples of bacterial selectable markersare the dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol, or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors of the present invention can contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Non-limiting examples of bacterial origins ofreplication are P15A on or the origins of replication of plasmidspBR322, pUC19, pACYC177 (which plasmid has the P15A ori), or pACYC184permitting replication in E. coli, and pUB110, pE194, or pTA1060,permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (See,e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433 [1978]).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors include,but are not limited to p3×FLAG™ expression vectors (Sigma-Aldrich),which include a CMV promoter and hGH polyadenylation site for expressionin mammalian host cells and a pBR322 origin of replication andampicillin resistance markers for amplification in E. coli. Othercommercially available suitable expression vectors include but are notlimited to the pBluescriptII SK(−) and pBK-CMV vectors (Stratagene), andplasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (See, Lathe et al., Gene 57:193-201 [1987]).

The skilled person will appreciate that, upon production of an enzyme,in particular, depending upon the cell line used and the particularamino acid sequence of the enzyme, post-translational modifications mayoccur. For example, such post-translational modifications may includethe cleavage of certain leader sequences, the addition of various sugarmoieties in various glycosylation and phosphorylation patterns,deamidation, oxidation, disulfide bond scrambling, isomerisation,C-terminal lysine clipping, and N-terminal glutamine cyclisation. Thepresent invention encompasses the use of engineered carboxyesteraseenzymes that have been subjected to, or have undergone, one or morepost-translational modifications. Thus, the engineered carboxyesterasesof the invention includes one which has undergone a post-translationalmodification, such as described herein.

Deamidation is an enzymatic reaction primarily converting asparagine (N)to iso-aspartic acid (iso-aspartate) and aspartic acid (aspartate) (D)at approximately 3:1 ratio. This deamidation reaction is, therefore,related to isomerization of aspartate (D) to iso-aspartate. Thedeamidation of asparagine and the isomerisation of aspartate, bothinvolve the intermediate succinimide. To a much lesser degree,deamidation can occur with glutamine residues in a similar manner.

Oxidation can occur during production and storage (i.e., in the presenceof oxidizing conditions) and results in a covalent modification of aprotein, induced either directly by reactive oxygen species, orindirectly by reaction with secondary by-products of oxidative stress.Oxidation happens primarily with methionine residues, but may occur attryptophan and free cysteine residues.

Disulfide bond scrambling can occur during production and basic storageconditions. Under certain circumstances, disulfide bonds can break orform incorrectly, resulting in unpaired cysteine residues (—SH). Thesefree (unpaired) sulfhydryls (—SH) can promote shuffling.

N-terminal glutamine (Q) and glutamate (glutamic acid) (E) in theengineered carboxyesterases are likely to form pyroglutamate (pGlu) viacyclization. Most pGlu formation happens in manufacturing, but it can beformed non-enzymatically, depending upon pH and temperature ofprocessing and storage conditions.

C-terminal lysine clipping is an enzymatic reaction catalyzed bycarboxypeptidases, and is commonly observed in enzymes. Variants of thisprocess include removal of lysine from the enzymes from the recombinanthost cell.

In the present invention, the post-translational modifications andchanges in primary amino acid sequence described above do not result insignificant changes in the activity of the engineered carboxyesteraseenzymes.

Host Cells for Expression of Carboxyesterase Polypeptides

In another aspect, the present invention provides a host cell comprisinga polynucleotide encoding an improved carboxyesterase polypeptide of thepresent invention, the polynucleotide being operatively linked to one ormore control sequences for expression of the carboxyesterase enzyme inthe host cell. Host cells for use in expressing the carboxyesterasepolypeptides encoded by the expression vectors of the present inventionare well known in the art and include but are not limited to, bacterialcells, such as E. coli, Geobacillus stearothermophilus, Lactobacilluskefir, Lactobacillus brevis, Lactobacillus minor, Mycobacteriumtuberculosis, Streptomyces and Salmonella typhimurium cells; fungalcells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichiapastoris (ATCC Accession No. 201178)); insect cells such as DrosophilaS2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293,and Bowes melanoma cells; and plant cells. Appropriate culture media andgrowth conditions for the above-described host cells are well known inthe art.

Polynucleotides for expression of the carboxyesterase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

Escherichia coli W3110 is a host strain that finds use in the presentinvention, although it is not intended that the present invention belimited to this specific host strain. The expression vector was createdby operatively linking a polynucleotide encoding an improvedcarboxyesterase into the plasmid pCK110900 operatively linked to the lacpromoter under control of the lad repressor. The expression vector alsocontained the P15a origin of replication and the chloramphenicolresistance gene. Cells containing the subject polynucleotide inEscherichia coli W3110 can be isolated by subjecting the cells tochloramphenicol selection.

Methods of Generating Engineered Carboxyesterase Polypeptides.

In some embodiments, to make the improved carboxyesterasepolynucleotides and polypeptides of the present invention, thenaturally-occurring carboxyesterase enzyme that catalyzes the amidationreaction is obtained (or derived) from T. fusca. In some embodiments,the parent polynucleotide sequence is codon optimized to enhanceexpression of the carboxyesterase in a specified host cell. As anillustration, the parental polynucleotide sequence encoding thewild-type carboxyesterase polypeptide of T. fusca was constructed fromoligonucleotides prepared based upon the known polypeptide sequence ofT. fusca carboxyesterase sequence available in Genbank database (Genbankaccession no. WP_011292850.1). The parental polynucleotide sequence,designated as SEQ ID NO: 1, was codon optimized for expression in E.coli and the codon-optimized polynucleotide cloned into an expressionvector, placing the expression of the carboxyesterase gene under thecontrol of the lac promoter and lacI repressor gene. Clones expressingthe active carboxyesterase in E. coli were identified and the genessequenced to confirm their identity. The codon-optimized polynucleotidesequence designated SEQ ID NO: 1 was the parent sequence utilized as thestarting point for most experiments and library construction ofengineered carboxyesterases evolved from the original wild-typecarboxyesterase.

In other embodiments, to make the improved carboxyesterasepolynucleotides and polypeptides of the present invention, thenaturally-occurring carboxyesterase enzyme that catalyzes the amidationreaction is obtained (or derived) from G. stearothermophilus. In someembodiments, the parent polynucleotide sequence is codon optimized toenhance expression of the carboxyesterase in a specified host cell. Asan illustration, the parental polynucleotide sequence encoding thewild-type carboxyesterase polypeptide of G. stearothermophilus wasconstructed from oligonucleotides prepared based upon the knownpolypeptide sequence of G. stearothermophilus carboxyesterase sequenceavailable in Genbank database (Genbank accession no. WP_033015113). Theparental polynucleotide sequence, designated as SEQ ID NO: 137, wascodon optimized for expression in E. coli and the codon-optimizedpolynucleotide cloned into an expression vector, placing the expressionof the carboxyesterase gene under the control of the lac promoter andlad repressor gene. Clones expressing the active carboxyesterase in E.coli were identified and the genes sequenced to confirm their identity.The polynucleotide sequence designated SEQ ID NO: 137 was the parentsequence utilized as the starting point for most experiments and libraryconstruction of engineered carboxyesterases evolved from the originalwild-type carboxyesterase.

In some embodiments, to make the improved carboxyesterasepolynucleotides and polypeptides of the present invention, thenaturally-occurring carboxyesterase enzyme that catalyzes the amidationreaction was obtained or derived from M. tuberculosis. In someembodiments, the parent polynucleotide sequence was codon optimized toenhance expression of the carboxyesterase in a specified host cell. Asan illustration, the parental polynucleotide sequence encoding thewild-type carboxyesterase polypeptide of M. tuberculosis was constructedfrom oligonucleotides prepared based upon the known polypeptide sequenceof M. tuberculosis carboxyesterase sequence available in Genbankdatabase (Genbank accession no. WP_003407276). The parentalpolynucleotide sequence, designated as SEQ ID NO: 139, was codonoptimized for expression in E. coli and the codon-optimizedpolynucleotide cloned into an expression vector, placing the expressionof the carboxyesterase gene under the control of the lac promoter andlad repressor gene. Clones expressing the active carboxyesterase in E.coli were identified and the genes sequenced to confirm their identity.The codon-optimized polynucleotide sequence designated SEQ ID NO: 139,was the parent sequence utilized as the starting point for mostexperiments and library construction of engineered carboxyesterasesevolved from the original wild-type carboxyesterase, as describedherein.

In some embodiments, engineered carboxyesterases are obtained bysubjecting the polynucleotide encoding the naturally occurringcarboxyesterase or a codon-optimized version of the polynucleotideencoding naturally-occurring carboxyesterase to mutagenesis and/ordirected evolution methods, as discussed above. Mutagenesis may beperformed in accordance with any of the techniques known in the art,including random and site-specific mutagenesis. Directed evolution canbe performed with any of the techniques known in the art to screen forimproved promoter variants including shuffling. Mutagenesis and directedevolution methods are well known in the art (See, e.g., U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905,6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674,6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053,6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204,6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740,6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186,6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547,6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542,6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647,6,483,011, 6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602,6,506,603, 6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746,6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430,6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001,6,902,922, 6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312,7,058,515, 7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387,7,421,347, 7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502,7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598,7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477,7,873,499, 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988,8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580,8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066,8,768,871, 9,593,326, 9,665,694, 9,684,771, and all related non-UScounterparts; Ling et al., Anal. Biochem., 254(2):157-78 [1997]; Dale etal., Meth. Mol. Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet.,19:423-462 [1985]; Botstein et al., Science, 229:1193-1201 [1985];Carter, Biochem. J., 237:1-7 [1986]; Kramer et al., Cell, 38:879-887[1984]; Wells et al., Gene, 34:315-323 [1985]; Minshull et al., Curr.Op. Chem. Biol., 3:284-290 [1999]; Christians et al., Nat. Biotechnol.,17:259-264 [1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri,et al., Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat.Acad. Sci. U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat.Biotechnol., 14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994];Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625;WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO2009/152336. It is not intended that the present invention be limited toany particular methods, as various methods find use in the art.

In some embodiments, where the improved enzyme property desired isthermal stability, enzyme activity may be measured after subjecting theenzyme preparations to a defined temperature and measuring the amount ofenzyme activity remaining after heat treatments. Clones containing apolynucleotide encoding a carboxyesterase are then isolated, sequencedto identify the nucleotide sequence changes (if any), and used toexpress the enzyme in a host cell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis (e.g., using theclassical phosphoramidite method described by Beaucage et al., Tet.Lett., 22:1859-69 [1981], or the method described by Matthes et al.,EMBO J., 3:801-05 [1984], as it is typically practiced in automatedsynthetic methods). According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors. In addition, essentially any nucleic acid can be obtained fromany of a variety of commercial sources (e.g., The Midland CertifiedReagent Company, Midland, Tex., The Great American Gene Company, Ramona,Calif., ExpressGen Inc. Chicago, Ill., Operon Technologies Inc.,Alameda, Calif., and many others).

Engineered carboxyesterase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well-known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B(Sigma-Aldrich).

Chromatographic techniques for isolation of the carboxyesterasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography (HPLC), ion exchange chromatography,gel electrophoresis, and affinity chromatography. Conditions forpurifying a particular enzyme will depend, in part, on factors such asnet charge, hydrophobicity, hydrophilicity, molecular weight, molecularshape, etc., and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved carboxyesterase enzymes. For affinity chromatographypurification, any antibody which specifically binds the carboxyesterasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with the carboxyesterase. The carboxyesterasepolypeptide may be attached to a suitable carrier, such as BSA, by meansof a side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacillus Calmette Guerin) and Corynebacterium parvum.

The carboxyesterases may be prepared and used in the form of cellsexpressing the enzymes, as crude extracts, or as isolated or purifiedpreparations. The carboxyesterases may be prepared as lyophilizates, inpowder form (e.g., acetone powders), or prepared as enzyme solutions. Insome embodiments, the carboxyesterases can be in the form ofsubstantially pure preparations.

In some embodiments, the carboxyesterase polypeptides can be attached toa solid substrate. The substrate can be a solid phase, surface, and/ormembrane. A solid support can be composed of organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of the substrate can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation. A plurality of supports can be configured on an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments.

Methods of Using the Engineered Carboxyesterase Enzymes and CompoundsPrepared Therewith

Whole cells transformed with gene(s) encoding the engineeredcarboxyesterase enzyme and/or the optional cofactor regenerationenzymes, or cell extracts and/or lysates thereof, may be employed in avariety of different forms, including solid (e.g., lyophilized,spray-dried, and the like) or semisolid (e.g., a crude paste).

The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like), followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde or immobilization to asolid phase (e.g., Eupergit C, and the like).

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a prechilled lyophilizationchamber, followed by the application of a vacuum. After the removal ofwater from the samples, the temperature is typically raised to 4° C. fortwo hours before release of the vacuum and retrieval of the lyophilizedsamples.

The quantities of reactants used in the amidation reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of carboxyesterase substrate employed. Thefollowing guidelines can be used to determine the amounts ofcarboxyesterase, and/or amine. Generally, ester and amine substrates canbe employed at a concentration of about 5 to 200 grams/liter using fromabout 50 mg to about 5 g of carboxyesterase.

Those having ordinary skill in the art will readily understand how tovary these quantities to tailor them to the desired level ofproductivity and scale of production.

The order of addition of reactants is not critical. The reactants may beadded together at the same time to a solvent (e.g., monophasic solvent,biphasic aqueous co-solvent system, and the like), or alternatively,some of the reactants may be added separately, and some together atdifferent time points. For example, the carboxyesterase and thecarboxyesterase substrate may be added first to the solvent.

For improved mixing efficiency when an aqueous co-solvent system isused, the carboxyesterase and amine may be added and mixed into theaqueous phase first. The organic phase may then be added and mixed in,followed by addition of the carboxyesterase substrate. Alternatively,the carboxyesterase substrate may be premixed in the organic phase,prior to addition to the aqueous phase

Suitable conditions for carrying out the carboxyesterase-catalyzedamidation reactions described herein include a wide variety ofconditions which can be readily optimized by routine experimentationthat includes, but is not limited to, contacting the engineeredcarboxyesterase enzyme and substrates at an experimental pH andtemperature and detecting product, for example, using the methodsdescribed in the Examples provided herein.

The carboxyesterase catalyzed amidation is typically carried out at atemperature in the range of from about 15° C. to about 75° C. For someembodiments, the reaction is carried out at a temperature in the rangeof from about 20° C. to about 55° C. In still other embodiments, it iscarried out at a temperature in the range of from about 20° C. to about45° C. The reaction may also be carried out under ambient conditions.

The amidation reaction is generally allowed to proceed until essentiallycomplete, or near complete, coupling of substrates is obtained. Amideformation (product) can be monitored using known methods by detectingsubstrates and/or product. Suitable methods include, but are not limitedto, gas chromatography, HPLC, and the like. Conversion yields of theamide product generated in the reaction mixture are generally greaterthan about 50%, may also be greater than about 60%, may also be greaterthan about 70%, may also be greater than about 80%, may also be greaterthan 90%, and are often greater than about 97%.

EXAMPLES

Various features and embodiments of the present invention areillustrated in the following representative examples, which are intendedto be illustrative, and not limiting.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and 1 (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);° C. (degrees Centigrade); RT (room temperature); MWD (multiplewavelength detector); CDS (coding sequence); DNA (deoxyribonucleicacid); RNA (ribonucleic acid); RP-HPLC (reversed-phased high performanceliquid chromatography); FIOP (fold improvement over positive control);HTP (high throughput); LB (Luria broth); TFA (trifluoroacetic acid);MeCN (acetonitrile); TEoA (triethanolamine); THF (tetrahydrofuran);Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Millipore (Millipore,Corp., Billerica Mass.); Difco (Difco Laboratories, BD DiagnosticSystems, Detroit, Mich.); Daicel (Daicel, West Chester, Pa.); Genetix(Genetix USA, Inc., Beaverton, Oreg.); Molecular Devices (MolecularDevices, LLC, Sunnyvale, Calif.); Applied Biosystems (AppliedBiosystems, part of Life Technologies, Corp., Grand Island, N.Y.),Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); ThermoScientific (part of Thermo Fisher Scientific, Waltham, Mass.); Corning(Corning, Inc., Palo Alto, Calif.); and Bio-Rad (Bio-Rad Laboratories,Hercules, Calif.); Phenomenex (Phenomenex, Inc., Torrence, Calif.);Epicentre (Epicentre, Madison, Wis.).

Example 1 Wild-type Carboxyesterase Gene Acquisition and Construction ofExpression Vectors

This Example describes the acquisition of the codon optimizedrecombinant polynucleotides encoding wild-type carboxyesterases (SEQ IDNOs: 2, 138, and 140) from which genes encoding engineeredcarboxyesterases in the following examples were derived, and expressionvectors and host cells suitable for such engineering.

The codon optimized versions of the wild-type genes (SEQ ID NO: 1, 137,and 139) encoding the wild-type carboxyesterases (SEQ ID NO: 2, 138, and140) of T. fusca, G. stearothermophilus, and M. tuberculosis,respectively, were synthesized for expression in E. coli. The codonoptimized gene was cloned into expression vector pCK11 0900 (See, e.g.,U.S. Pat. No. 9,714,437 and U.S. Pat. Appln. Publn. No. 2006/0195947,both of which are incorporated herein by reference in their entiretiesand for all purposes), under the control of a lac promoter. Theexpression vector also contained the P15a origin of replication and thechloramphenicol resistance gene. These sequence-verified vectors weretransformed into a E. coli W3110 strain for expression. Thepolynucleotides (odd numbered SEQ ID NOs: 1-135) encoding the engineeredcarboxyesterases (even numbered SEQ ID NOs: 2-136) of the presentinvention were likewise cloned into vector pCK11 0900 for expression ina derivative of E. coli W3110 strain. Directed evolution techniquesgenerally known to those skilled in the art were used to generate thelibraries of the engineered carboxyesterases.

Example 2 HTP Production and Analysis of Wild-Type CarboxyesterasePolypeptides

HTP lysates were prepared by taking the codon-optimized carboxyesterasegenes (described in Example 1) which were then transformed into E. coliW3110 and plated on Luria-Bertani (LB) agar medium containing 1% glucoseand 30 μg/mL chloramphenicol (CAM). After incubation for at least 16 hat 30° C., colonies were picked using a Q-botobotic colony picker(Genetix) into a 96-well shallow well microtiter plate containing 200 μLof LB, 1% glucose, and 30 μg/mL CAM. Cells were grown 18-20 h at 30° C.,with shaking at 200 rpm. Then, 20 μL of these cultures were thentransferred to 360 μL of Terrific Broth (TB) and 30 μg/mL CAM in a deepwell plate. After incubation at 30° C. with shaking at 250 rpm for 2.5 h(OD₆₀₀ 0.6-0.8), recombinant gene expression was induced by addingisopropyl thioglycoside (IPTG) to a final concentration of 1 mM. Theplates were then incubated at 30° C., with shaking at 250 rpm for 18-21h.

Cell cultures were pelleted at 3500×g for 20 mM, and the mediadiscarded. Cell pellets were lysed in 200 μL of 0.2 M TEoA, pH 7.5 with1 g/L lysozyme, 0.5 g/L polymixin B sulfate, and 0.5 μL OmniCleave™endonuclease (Epicentre) by shaking at RT for 2 h. Samples werecentrifuged at 3500×g for 20 min to clarify cellular debris, and thesupernatant was used to carry out the transformations described inExamples 3 and 4.

Example 3 Activity of Wild-Type Carboxyesterases on Substrate Set #1

The wild-type carboxyesterase polypeptides were generated as describedin Example 2. To analyze for amidation activity on substrate set #1(Table 3.1), 20 μL supernatant were added to a mixture of 10 μL aniline,10 μL ethyl acetate, 100 μL of 0.1 M sodium phosphate buffer, pH 7.0.Reactions were incubated at 20° C. and shaken at 300 rpm for 18 h.Samples were quenched by diluting 2-fold in MeCN. Analysis of reactionswas performed by RP-HPLC as described in Example 12.1.

TABLE 3.1 Substrate Sets Used to Evaluate Carboxyesterase AmidationActivity Products Set 1

Set 2

Set 3

Set 4

Set 5

Set 6

Set 7

Set 8

Set 9

Set 10

Set 11

Set 12

Set 13

Set 14

Set 15

Example 4 Activity of Wild-Type Carboxyesterases on Substrate Sets #2-7

Wild-type carboxyesterase polypeptides were generated according to themethods described in Example 2. To analyze for amidation activity onsubstrate set #2 (See, Table 3.1), 100 μL supernatant was added to amixture of 10 μL methyl phenylacetate, 10 μL n-butylamine, 10 μLdimethyl sulfoxide (DMSO), and 20 μL of 0.1 M sodium phosphate buffer,pH 7.0. Reactions were incubated at RT and shaken at 300 rpm for 18 h.Samples were quenched by diluting with an equal volume of MeCN. Analysisof substrates and products was performed by RP-HPLC using the methodsdescribed in Example 12.1.

To analyze for amidation activity on substrate sets #3-7 (See, Table3.1), 120 μL of a mixture of 75-100 g/L amine and 15 g/L ethyl benzoatein 0.1 M potassium phosphate buffer, pH 7.5, with or without 50%isopropanol, were added to 100 μL supernatant, produced as described inExample 2. Reactions were incubated at 55° C. and shaken at 400 rpm for18 h. Samples were quenched by diluting 3-fold in MeCN and thencentrifuged for 5-8 min at 4000×g. The resultant supernatant wasanalyzed by RP-HPLC using the methods described in Example 12.2. In oneexperiment using substrate set #3, 120 μL of a mixture of 5 g/Lisobutylamine and 15 g/L ethyl benzoate in 0.1 M potassium phosphatebuffer, pH 7.5, was added to 100 μL supernatant, produced as describedin Example 2. The results for all of these substrate sets are providedin Table 4.1 with the substrate sets indicated by “reaction” numbers(i.e., “R1,” “R2,” “R3,” etc.).

TABLE 4.1 Solvent Tolerance and Substrate Scoping of Wild-typeCarboxyesterases (SEQ ID NOs: 2, 138, and 140)¹ R1 R2 R3 R4 R5 R6 R7Conditions (KPi Buffer, pH 7.5) T. fusca ‡ † +++++ +++ +++ G.stearothemophilus ## +++++ # + † M. tuberculosis # ++++ + +++ ++++Conditions (KPi Buffer, pH 7.5/ IPA (50:50 v/v %)) T. fusca † ++++ G.stearothemophilus ## # ++ M. tuberculosis ## + + +++ ¹Levels ofincreased activity were determined relative to the reference peak areaof the negative control powder. In this Table, “+” = >0, <20; “++”= >20, <50; “+++” = >50, <100; “++++” = >100, <500; “+++++” = >500,<1000; “#” = >1000, <2000; “##” = >2000, <5000; “†” = >5000, <10000;“‡”= >10000, <15000.

Example 5 Production of Wild-Type Carboxyesterase Shake Flask Powders

The wild-type carboxyesterases (SEQ ID NOs: 2, 138, and 140) wereproduced in shake flasks for further characterization. The E. colitransformants containing the plasmid encoding WT carboxyesterases weregrown on Luria-Bertani (LB) agar medium containing 1% glucose and 30Kg/mL chloramphenicol (CAM). After incubation for at least 16 h at 30°C., single colonies were picked into 6 mL of LB, 1% glucose, and 30μg/mL CAM. Cells were grown 18-20 h at 30° C., with shaking at 250 rpm.This culture was then transferred into Terrific Broth (TB) and 30 μg/mLCAM at a final OD₆₀₀ of 0.2 and a final volume of 250 mL. Afterincubation of the flasks at 25° C. or 30° C. with shaking at 250 rpm for2.5 h (OD₆₀₀ 0.6-0.8), recombinant gene expression was induced byisopropyl thioglycoside (IPTG) to a final concentration of 1 mM. Theflask was then incubated at 30° C. with shaking at 250 rpm for 18-21 h.Cells were pelleted at 3500×g for 20 min, and the supernatant wasdiscarded. The cell pellet was washed 3× in 30 mL ice cold 50 mM sodiumphosphate pH 7.5, resuspended in 12 mL of the same buffer, and lysedusing a cell disruptor at 18-20 kpsi. Lysates were clarified at 10000×gfor 30 min, and clarified supernatants were lyophilized to an off-whitepowder.

TABLE 5.1 Wild-type Carboxyesterase (SEQ ID NO: 2, 138, and 140) GrowthEvaluation SEQ Growth Growth Harvest Harvest ²Pellet Pellet ³PowderPowder ID NO: OD₆₀₀ ¹ OD₆₀₀ OD₆₀₀ OD₆₀₀ Mass Mass Mass Mass Shake Flask25° C. 30° C. 25° C. 30° C. 25° C. 30° C. 25° C. 30° C. Neg. Ctrl. — ++++++ ++ ++ ++++ +++ + ++ T. fusca  2 ++++ ++++ +++ ++++ +++ ++++ ++ ++ G.stearothermophilus 138 ++++ ++++ + N/A ++ N/A ++ N/A M. tuberculosis 140++++ ++++ +++ +++ ++++ ++++ +++ +++ ¹OD 600 rankings as follows (“+”= >10, <15: “++” = >15, <20: “+++” = >20, <25; “++++” = >25, <30); ²Masspellet rankings as follows (“+” = >3 g, <4 g; “++” = >4 g, <5 g; “+++”= >5 g, <6 g; “++++” = >6 g, <7 g); ³Mass of powder ranking as follows(“+” = >0.2 g, <0.5 g; “++” = >0.5 g, <1.0 g; “+++” = >1.0 g, <1.5 g)

Example 6 Solvent Tolerance Evaluation of Wild-Type CarboxyesterasePolypeptides

In this Example, experiments conducted to determine the solventtolerance of the wild-type carboxyesterases are described. In theseexperiments, the tolerance to organic solvent of the lyophilized shakeflask powders prepared in Example 5 was determined, by testing activityon substrate sets #3 and #5. Reactions were conducted in a 96 well plate(reaction volume 220 μL) with 10 g/L enzyme powder, 30-34 g/L amine, 43g/L ester, and 10% DMSO, or 50% isopropanol, or 15-25% MeCN or 25% THFin 0.1 M potassium phosphate buffer, pH 7.5. Reactions were heated to50° C. with shaking at 400 rpm for 18-21 h. Reactions were quenched bydiluting 3-fold in MeCN. Reaction samples were analyzed by RP-HPLC usingmethods described in Example 12.2.

TABLE 6.1 Solvent Tolerance Evaluation of Wild-type Carboxyesterases(SEQ ID NO: 2, 138, and 140) Substrate G. M. Set Co-solvent T. fuscastearothemophilus tuberculosis #3 Buffer Only +++++ ++ ++ MeCN 15% ++ ++++ 25% ++ ++ ++ THF 15% ++ − ++ 25% ++ ++ ++ DMSO 15% ++++ + − 25% +++++ ++ #5 Buffer Only ++ − − MeCN 15% + + ++ 25% + + − THF 15% ++ − −25% + − − DMSO 15% ++ − − 25% ++ − − ¹Levels of increased activity weredetermined relative to the reference negative control powder (Table 5.1)and defined as follows: “−” = no activity; “+” >1-fold activity;“++” >1-fold activity, <2-fold activity; “+++” >2-fold, <3-foldactivity; “++++” >4-fold, <5-fold activity; “+++++” >than 5-foldactivity.

Example 7 Production and Analysis of Engineered T. fusca CarboxyesterasePolypeptide Libraries

Plasmid libraries containing evolved T. fusca carboxyesterase genes weretransformed in E. coli according to the methods described in Example 1,and produced following the methods described in Example 2. The celllysates were used to carry out the activity assessments described inExamples 8 and 9.

Example 8 Amidation Activity of Engineered T. fusca CarboxyesterasePolypeptides on Substrate Sets #3, 8-12

T. fusca carboxyesterase variants were generated according to Example 7.To analyze for amidation activity on substrate set #3 (See, Table 3.1),20 μL of supernatant produced as described in Example 7, were added to amixture of 25 μL MeCN with 3 M ethyl benzoate, 110 μL of 0.6 Misobutylamine in 0.2 M TEoA (pH adjusted to 9.5), and 65 μL 0.2 M TEoA,pH 8.5. Reactions were incubated at 50° C. and shaken at 300 rpm for 18h. Samples were quenched by diluting 4-fold in MeCN. Analysis ofreactions was performed by RP-HPLC using the methods described inExample 12.2.

To evaluate for amidation activity on substrate sets #3, 8-12 (See,Table 3.1), 20-50 μL supernatant produced as described in Example 7,were added to a mixture of 25 μL MeCN with 2-3.2 M amine, 110 μL of0.4-0.6 M ester suspension in 0.2 M TEoA (pH adjusted to 8), and 35-65μL 0.2 M TEoA, pH 8.5-9.0. Reactions were incubated at 50° C. and shakenat 300 rpm for 16-18 h. Samples were quenched by diluting 4-fold inMeCN. Analysis of reactions was performed by RP-HPLC, as described inExamples 13.3, 13.4, and 13.5. In one experiment using substrate set #8,50 μL supernatant produced as described in Example 7, were added to amixture of 25 μL MeCN with 3 M glycine methyl ester, 110 μL of 0.6 Maniline in 0.2 M TEoA (pH adjusted to 8), and 35 μL 0.2 M TEoA, pH 8.5.Reactions were incubated at 50° C. and shaken at 300 rpm for 18 h.Samples were quenched by diluting 4-fold in MeCN. Analysis of reactionswas performed by RP-HPLC using the methods described in Example 12.3.The amino acid substitutions of these variants are indicated relative toSEQ ID NO: 2.

TABLE 8.1 Amidation¹ by T. fusca Carboxyesterase Variants Relative toSEQ ID NO: 2 on Substrate Sets 3, 8, 9, 10, 11, and 12 Variant SEQ IDAmino Acid Set Set Set Set Set Set No. NO: (nt/aa) Mutations 3 8 9 10 1112 1 A108W *** 2 3/4 L282Q *** 3 5/6 L282A *** * * 4 7/8 L282T *** ****** 5 A108K *** 6 9/10 A285L ** * 7 I69F ** 8 11/12 L282C ** * 9 L282W** 10 L282R ** 11 F323Y ** 12 I69W * 13 13/14 A108G * *** 14 I69Y * 15T110A * 16 A108Q * 17 A285P * 18 T373G * 19 15/16 N215R * * 20 T317P *21 I69L * 22 N215K * * 23 A108R * 24 17/18 L281P * ***** 25 19/20N215W * * 26 L209P * 27 P283K * 28 F377Y * 29 A213P * 30 G70W * 31A249V/F284P * 32 21/22 G212P * * 33 A186G * 34 I372A/V376A * 35 A71R * *36 Y320S * 37 135/ I372L * 136 38 A71Y * 39 A108S * 40 S115T * 41 A71G *42 A71F * 43 S190H * 44 23/24 V280G * ***** 45 P64W * 46 I69V * 47L209E * 48 P117A * 49 A68L * 50 P64I * 51 L209S * 52 Y65W * 53 25/26N215P * * 54 P63A * 55 S279L/V280G/ * L282M 56 T110S * 57 S115H * 58V376M * 59 V376L * 60 G70L * 61 G214T * 62 G212A * 63 V376A * 64 F284T *65 P405D * 66 S185T * 67 G70R * 68 V280E * 69 27/28 P283D * ***** 70Y320S/I372A/ * V376G 71 G70T * 72 P64E * 73 Y320F * 74 R428V * 75Y320A * 76 R321S * 77 W271K * 78 W271P * 79 Y65T * 80 V376G * 81 F377W *82 V118I * 83 F323R * 84 A217W * 85 G214K * 86 A429L * 87 T110H * 88T110P * 89 R153L * 90 29/30 Y320G * *** 91 Y320S/V376G/ F377V * 92G114H * 93 V280S * 94 31/32 R321L * *** 95 Y320S/F323S/ * I372A 96T224I/P268S/ * I372F 97 V118N * 98 A217V * 99 A71P * 100 L209V * 101Y320G/F323S * 102 W271L * 103 P420G * 104 33/34 W271T * * 105 P283C *106 35/36 P63R * * 107 37/38 Y320W * * 108 39/40 S190K * * 109 R126C**** 110 L282S * 111 F284C ***** 112 F284V * 113 A269N * 114 W271A **115 P283T **** 116 A71H/Q263R * 117 41/42 N215R/W271R * * 118 A217G *119 A213S ** 120 G277M *** 121 L281V *** 122 43/44 P283V **** 123D427A * 124 P63Y * 125 V270I/V470M * 126 A269V * 127 S279G **** 128A217S * 129 45/46 A68P * * 130 P63T * 131 A285M * * 132 47/48 P283Y***** 133 L311I * 134 S279C **** 135 V270I * 136 V270R ** 137 T317C **138 W271S * * 139 S279V **** 140 A217R * 141 S190L * 142 P64A * 143A71V * 144 P117F * 145 49/50 E184S/A249T ***** 146 51/52 F284T/P438T***** 147 L209G * 148 G278H *** 149 L324A **** 150 S190M * 151 P64G *152 A276F **** 153 P64V * 154 P64T * 155 P66N * 156 A217L * 157 I69H *158 M216P * 159 A213N * 160 A217R/A231V * 161 A213T/W271K * 162V118G/A349V * 163 N215M * 164 A188G * 165 S190Q * 166 T39M/F323I * 167G278S **** 168 V118N/A269T * 169 A213C * 170 Y65S * 171 P283R/A429V *172 A213V * 173 53/54 A213L * * 174 A186C * 175 E184F * 176 A213Q * 177I104Q/A429V * 178 A217P * 179 N111W * 180 55/56 F377L ** 181 57/58E184G * ***** 182 G214V * 183 F323C * 184 59/60 R153H/N215P * * 185W164R/W271T ** 186 G212R * 187 P286V ** 188 F323I * 189 N111M * 190I69G * 191 61/62 G214L * 192 G212S * 193 W271Q/A416V * 194 63/64S190W * * 195 Q210T * 196 G114Q * 197 N111V * 198 Y119S * 199 N111L *200 A213E * 201 S211I * 202 A186T * 203 F109G/P117M * 204 Y119G * 205S211V * 206 L281Y/D374N * 207 E184P ***** 208 Y119P * 209 A213R/5345G *210 W103T/P147S * 211 W103R * 212 N111S * 213 W103P * 214 Q210W * 215I104P * 216 S211L * 217 S190R * 218 G183P * 219 Q210P * 220 A188E * 221H105L * 222 G107P * 223 S113P * 224 G114A * 225 65/66 F77S/E184G *****226 S279E **** 227 G107D/S185W ** 228 S211R * 229 S185A * 230 A186R *231 G187P * 232 A186P * 233 R62H/P117G * 234 N111R * 235 S115V * 236G107L * 237 G1075 * 238 Y65G * 239 E184Y * 240 G174D/L282V * ¹Levels ofincreased activity were determined relative to the reference polypeptideand defined as follows: <1.5×; “*” = >1.5×, <3.5×; “**” = >3.5×, <5.5×;“***” = >5.5×, <7.5×; “****” = >7.5×, <9.5×; “*****” = >9.5×, <10.5×.

Example 9 Evaluation of Shake Flask Powders of Engineered T. fuscaCarboxyesterase Polypeptides

Powders of evolved T. fusca carboxyesterases were prepared in shakeflask scale quantities following the methods described in Example 5. Theamidation activity of the lyophilized shake flask powders was assessedby testing their activity on substrate set #3. First, 1.0 mL reactionmixtures were prepared with 10 g/L enzyme powder, 0.3 M isobutylamine,0.3 M ethyl benzoate in toluene, followed by addition of 20 μL of 0.2 MTEoA buffer, pH 8.5. Reactions were heated to 50° C. with shaking at 500rpm for 60 h. Reactions were quenched by diluting 100 μL reactionmixture into 1.4 mL acetone. Reaction samples were analyzed by RP-HPLCusing the methods provided in Table 12.2.

TABLE 9.1 Amide Formation by T. fusca Carboxyesterase Variants AmideFormation (FIOP)¹ Variant No.: Relative to SEQ ID NO: 2  4 ++++  3 ++++12 +++ 31 +++ 28 ++ 24 ++ 37 ++ ¹Levels of increased activity orselectivity were determined relative to the reference polypeptide of SEQID NO: 2 and defined as follows: “++” > than 1.2-fold but less than2.5-fold increase; “+++” > than 2.5-fold but less than 5-fold; “++++” >than 5-fold but less than 10-fold.

Example 10 Production and Analysis of Combinatorial T. fuscaCarboxyesterase Libraries

Plasmid libraries obtained through combinatorial shuffling on a T. fuscacarboxyesterase variant (SEQ ID NO: 8) were transformed in to E. coliW3110 according to the methods described in Example 1. The HTP lysatesproduced according to the methods described in Example 2, were used tocarry out the activity assessments described in Example 11.

Example 11 Activity of Engineered T. fusca Carboxyesterase Polypeptideson Selected Substrate Sets 3, 5, 8, 12-15

T. fusca carboxyesterase variants generated from combinatorial librarieswere analyzed for amidation activity on substrate sets #3, 5, 8, and12-15 (See, Table 3.1). To analyze for amidation activity on substrateset #3 (See, Table 3.1), 10 μL supernatant produced as described inExample 7, and were added to a mixture of 25 μL MeCN with 3 M ethylbenzoate, 110 μL of 0.6 M isobutylamine in 0.2 M TEoA (pH adjusted to8), and 75 μL 0.2 M TEoA, pH 8.5. Reactions were incubated at 50° C. andshaken at 300 rpm for 18 h. Samples were quenched by diluting 4-fold inMeCN. To analyze for amidation activity on substrate sets #5, 8, and12-15, 20-50 μL supernatant were produced as described in Example 7, andadded to a mixture of 25 μL MeCN with 2-3.2 M amine, 110 μL of 0.4-0.6 Mester suspension in 0.2 M TEoA (pH adjusted to 8), and 35-65 μL 0.2 MTEoA, pH 8.5-9. Reactions were incubated at 50° C. and shaken at 300 rpmfor 16-18 h. Samples were quenched by diluting 4-fold in MeCN. Analysisof reactions was performed by RP-HPLC, using the methods described inExamples 12.2 (Set 3), 12.3 (Set 8), 12.5 (Set 12, and 13), 12.6 (Set14), and 12.7 (Set 5 and 15). In one experiment using substrate set #8,20 μL supernatant produced as described in Example 7 were added to amixture of 25 μL MeCN with 3 M glycine methyl ester, 110 μL of 0.6 Maniline in 0.2 M TEoA (pH adjusted to 9.5), and 65 μL 0.2 M TEoA, pH8.5. Reactions were incubated at 50° C. and shaken at 300 rpm for 18 h.Samples were quenched by diluting 4-fold in MeCN. Analysis of reactionswas performed by RP-HPLC, using the methods describe in Example 12.3.

TABLE 11.1 Amidation¹ by T. fusca Carboxyesterase Variants Relative toSEQ ID NO: 8 on Substrate Sets 3, 5, 8, and 12-15 SEQ ID Variant NO: No.(nt/aa) Amino Acid Mutations Set 3 Set 5 Set 8 Set 12 Set 13 Set 14 Set15 241 67/68 Y65W/I69L/I372L **** 242 Y65W/I69W/G70L/I372L *** 243I69W/G70L/G459R *** 244 I69W/G70L *** 245 G70L ** 246 69/70A68P/I69L/A343V/I372L * * * 247 I69L/G70L/P331Q/I372M * * 248 71/72I69L/W271Y/I372L * * * 249 73/74 I372L * * * * 250 75/76P63R/A108G * * * 251 R126C/I372L * 252 Y320W * * 253 I69L/I372L * 254I69W/G212A/A213L/N215R/ * V280G/L281P 255 I69W/G70L/I372M * 256 77/78Y65W/I69M/G70A/L281P/ * * * I372L 257 A108G * * * 258 R126C * 259 79/80I69W/I372M * * ** * 260 I69M/G70A/I372M * * * 261I69L/T282C/A343V/I372L * 262 P63R * * 263 81/82 V280G/A285L/I372L * * *264 A68P/I69L/W271Y * * 265 83/84 W271Y/A343V/I372L * * * * 266 85/86I69L * * * * 267 87/88 L281P/I372L * * * * 268 I69L/W271Y * 269I372M * * 270 89/90 P63R/Y65G/A108G * *** 271 A343V/I372L * 272 91/92A68P/W271Y/I372L ** ** 273 93/94 A108G/A285L * ** 274Y65W/G70L/I372M * * 275 I372L/A381L * ** 276 I69W/N215P *** 277 I69W***** * 278 A108G/V270E * * 279 W271Y/A343V/I372L/A381L ***** 280 P63Y *281 A108G/F377I **** 282 P63A * 283 A68P/I69L/T282C/V287I * 284T373G * * 285 I69W/T282A * 286 95/96 A108G/N215K * ***** 287N215P/I372L * * 288 Y65G/Y320W * 289 I69Y/A108G/L281P/A285P * 290P63R/A108G/Y320W/F323C * 291 I69L/N215P/W271Y/I372L ** 292 97/98 A68P** * * 293 P63Y/P268A/ * * A269N/V270I/ A429V 294 W271Y * 295W271Y/A343V ***** 296 A68P/I69W/N215P ***** 297R126C/E184S/A213S/I372L * 298 A108G/M189L/Y320W *** 299I69F/A108G/V270E/I372L/ ***** F377L 300 I69L/N215P/W271Y/ ***V280G/L281P/T282C 301  99/100 N215R/I372L * ** 302 P63R/F377I **** 303A108G/Y320W/F323I * 304 I372L/F377L ***** 305 N215P/W271Y/I372L *** 306I69L/N215P/A343V/I372L/ * A381L 307 R126C/N215P * 308 101/102N215K/L281P/A285L/I372L ** ** 309 A68P/F377L ** 310A68P/I69W/G214R/N215P/ * * W271Y 311 A68P/A108G/F377L ***** 312 F377L **313 103/104 P63T/N215R * * ** 314 A68P/I69W/M189I/A381L ** 315A68P/E184S * 316 105/106 I69W/N215R * ***** 317 I69L/N215P/W271Y/T282A** 318 I69W/A108S * 319 N215P/Y320G * * 320 N215R/V280G/L281P/ * *A285L/I372L 321 V181L/N215P * 322 107/108 P63A/N215R/A343V * * *** 323N215P/Y320G/I372L * 324 M189I/F377I *** 325 109/110 P63Y/G212P/N215R * *** 326 A108G/F377L *** 327 P63Y/N215P/A269N * * 328 I69W/N215K/A343V *329 A68P/I69W/M189E/G214R/ * N215P/W271Y/L281P/ T282G/A343V/A381L 330111/112 P63Y/G212P/N215R/ * ** P268A/A269N/A343V 331 I69F/N215K * *****332 G214R/N215P/W271Y *** 333 I69F/A285L/T373G * * 334N215R/A249T/V280G/ * * L281P/A285L/I372L 335 A68P/I69L/N215P * 336A68P/I69W/M189I/I372L ** 337 R126C/M189I/A285L/I372L ** 338 N215P * **339 A249T/F377L * 340 N215K/A285L/V445L * *** 341 113/114P63Y/N215R * * * 342 P63A/M189A * 343 N215K * *** 344 N215K/F323Y * *345 A285L/F323I * 346 A68P/I69W/M189I/W271Y * 347I69W/M189I/A343V/A381L * 348 P63Y/N215R/V2701/W271S * 349 N215R * * *350 I69W/M189E/I372L * 351 N215P/I372L/F377L * ***** 352A68P/N215P/W271Y/A343V/ * * I372L/A381L 353 N215R/Y320G * *** 354I69W/M189I/W271Y *** 355 I69L/M189I/V280G/T282G/ ***** A343V/I372L/A381L356 P63R/A108G/A285L/F377I ***** 357 I69W/M189E/W271Y/A343V * 358I69W/M189I * 359 A68P/G214R/N215P/W271Y/ * L281P/T282A/I372L 360M1891/Y320W/F377I **** 361 N215K/L281P/T373G * * 362 N215R/A285P ****363 M189I/V270E/A285L ** 364 P63R/Y65G/A108G/F377I ***** 365I69W/N215R/F323Y * * 366 M189Q ***** 367 M189I/N215P/A343V/I372L * 368M189Q/A343V ***** 369 P63R/Y65G/Y320W/F323I ** 370I69W/G214R/N215P/W271Y/ ***** I372L/F377Y/A381L 371A108G/T282A/A285L/F377L ***** 372 N215P/A381L * * 373A108G/M189I/T282A/A285L/ ** Y320W 374 T282A/A285L/Y320W/F323I ** 375P63R/A108G/T282A/A285L/ ***** F377L 376 I69W/G214R/W271Y/T282A ** 377M189Q/A381L **** 378 115/116 A68P/I69W/M189I/G214R/ ***** * * N215P 379Y65G/Y320W/F323I *** 380 117/118 N215K/T373G * **** 381I69W/M189Q/I372L/F377Y ***** 382 M189E/I372L/F377Y ***** 383 A108G/M189L** 384 A68P/I69W/M189E/A343V/ * A381L 385 I69W/M189E/G214R/A343V/ *****I372L 386 I69L/M189I/T282A * 387 A68P/M189I/W271Y/I372L * 388G214R/N215P/W271Y/F377Y * 389 119/120 N215K/I372L/F377L * ***** 390121/122 M1891/A343V ***** * 391 123/124 N215R/L281P/A285L/T373G *** *392 125/126 N215W/A285L/G346S * ** 393 G214R/W271Y * 394R126C/E184S/A213S/V280G/ ** L281P/A285L/Y320G 395 A68P/I69L/M189I/L281P/*** T282C/I372L/F377Y/A381L 396 A68P/M189I/A343V * 397I69L/M189I/W271Y/A343V/ ** A381L 398 M189L ** 399 P63Y/M189L * 400I69L/M189I ** 401 A68P/I69W/G214R/A343V * 402 N215W * * 403M189I/V280G/T282A * 404 N215W/A285P ** 405 M189I/N215K * 406A68P/I69L/M189I/G214R/ * N215P/W271Y 407 A68P/I69L/M189Q/W271Y/ **V280G/I372L/A381L 408 127/128 M189I ** * * 409 P63R/Y65G/T282A/A285L/ *Y320W/F323I 410 I69W/M189I/N215P/A343V * 411 A108G/M189L/F377I ** 412I69F/M189L ** 413 N215K/A285L/T317P * * 414 I69Y/T110A/N215R/L281P * 415I69L/N215R/A285P/T317P * 416 I69W/G214R/W271Y/A343V * 417A213S/N215P/Y320G * 418 I69Y/M189L/L281P/T373G * 419 N215W/T373G * * 420M1891/G214R/N215P/ * W271Y/T282G 421 P63R/Y65G/A108G/M189L * 422 129/130I69F/N215K/A269L/ ***** V270I/F377L 423 A68P/N215P/F377L **** 424N215P/F377L **** 425 I69L/M189I/G214R/W271Y/ ** L281P/T282A/A343V 426M189I/N215R/A249T/G277M * 427 G70L/G212P * 428 A68P/E184S/M189E ** 429M189Q/G214R ***** 430 M189Q/A343V/F377Y ***** 431 131/132M189Q/N215P/W271Y/ ***** L281P/T282C/F377Y 432 G214R/V280G/T282A/ **A343V/F377Y/A381L 433 A68P/I69L/M189E/G214R/ ** W271Y/V280G 434A68P/I69W/M189E/V280G/ ** L281P/T282A/I372L/F377Y 435A68P/I69L/M189E/G214R/ * I372L 436 I69L/M189E/W271Y/L281P/ * T282A 437I69L/M189Q/F377Y * 438 A68P/I69L/M189Q/G214R * 439A68P/I69W/M189I/G214R/ * F377Y/A381L 440 133/134 A108G/N215P/F377L *****441 I69F/N215R * *** 442 N215K/I372L * *** 443 V270E/F377L * 444A269L/V270E/L281P/ * I372L/F377L 445 M189I/V270E/I372L * 446A108G/A269L/V270E * ¹Levels of increased activity were determinedrelative to the reference polypeptide and defined as follows: <1.5×; “*”= >1.5×, <3.5×; “**” = >3.5×, <5.5×; “***” = >5.5×, <7.5×; “****”= >7.5×, <9.5×; “*****” = >9.5×, <10.5×.

Example 12 Analytical Detection of Produced Amides and PrecursorSubstrates

Data described in the above Examples were collected using analyticalmethods in Tables 12.1 through 12.7. The methods provided herein allfind use in analyzing the products from the T. fusca carboxyesterasevariants produced using the present invention. However, it is notintended that the present invention be limited to the methods describedherein for the analysis of the products provided herein and/or producedusing the methods provided herein. Indeed, any suitable method finds usein the present invention. Product peak elution was confirmed either byconfirmation with a commercially available standard or by LC/MS/MSanalysis.

TABLE 12.1 Analytical Method Instrument Agilent 1100 Series HPLC ColumnAgilent XDB C-18, 4.6 × 100 mm, 5 μm Mobile Phase Gradient I (20%Methanol; 80% Water) Isocratic Gradient Flow Rate 1.000 mL/min Run Time10.0 min Elution order Substrate Set #1-Aniline, acetanilide (1)Substrate Set #2-amide (2), methyl phenylacetate Column RT TemperatureInjection Volume 10 μL Detection UV 254 nm; Detector: MWD

TABLE 12.2 Analytical Method Instrument Agilent 1100 Series HPLC ColumnPhenomenex Luna, C-18 4.6 × 150 mm, 5 μm Gradient I (A = 0.1% TFA inwater, B = 0.1% TFA in MeCN) Time(min) % A Mobile Phase 0.000 100 7.000 5 Flow Rate 1.5 mL/min Run Time 8.000 min Elution order Substrate Set#3-isobutylbenzamide (3), ethyl benzoate Substrate Set#4-t-butylbenzamide (4), ethyl benzoate Substrate Set #6-t-butoxy-benzamide (6), ethyl benzoate Substrate Set#7-N′-t-butoxycarbonyl- benzhydrazide (7), ethyl benzoate ColumnTemperature 25° C. Injection Volume 10 μL Detection UV 254 nm; Detector:MWD

TABLE 12.3 Analytical Method Instrument Agilent 1100 Series HPLC ColumnPhenomenex Luna, C-18 4.6 × 150 mm, 5 μm Gradient I (A = 0.1% TFA inwater, B = 0.1% TFA in MeCN) Time(min) % A Mobile Phase 0.000 90 2.75045 3.150  5 Flow Rate 1.5 mL/min Run Time 5.000 min Elution orderSubstrate Set #8-aniline, (amide 8) Column 30° C. Temperature InjectionVolume 10 μL Detection UV 220 and 260 nm; Detector: MWD

TABLE 12.4 Analytical Method Instrument Agilent 1100 Series HPLC ColumnPhenomenex Luna, C-18 4.6 × 150 mm, 5 μm Gradient I (A = 0.1% TFA inwater, B = 0.1% TFA in MeCN) Time(min) % A Mobile Phase 0.000 90 2.75030 3.150  5 Flow Rate 1.5 mL/min Run Time 5.000 min Elution orderSubstrate Set #9-benzylamine, (amide 9) Column Temperature 30° C.Injection Volume 10 μL Detection UV 230 and 260 nm; Detector: MWD

TABLE 12.5 Analytical Method Instrument Agilent 1100 Series HPLC ColumnPhenomenex Luna, C-18 4.6 × 150 mm, 5 μm Gradient I (A = 0.1% TFA inwater, B = 0.1% TFA in MeCN) Time(min) % A Mobile Phase 0.000 95 1.10075 5.900 30 Flow Rate 1.5 mL/min Run Time 8.000 min Elution orderSubstrate Set #10-S-phenylethylamine, (amide 10), ethyl benzoateSubstrate Set #11-R-phenylethylamine, (amide 11), ethyl benzoateSubstrate Set #12-4-methylpiperidine, pyrazine ethyl ester, (amide 12)Substrate Set #13-S-phenylethylamine, pyrazine ethylester, (amide 13)Column Temperature 25° C Injection Volume 10 μL Detection UV 230 and 260nm; Detector: MWD

TABLE 12.6 Analytical Method Instrument Agilent 1100 Series HPLC ColumnPhenomenex Luna, C-18 4.6 × 150 mm, 5 μm Gradient I (A = 0.1% TFA inwater, B = 0.1% TFA in MeCN) Time(min) % A Mobile Phase 0.000 70  2.7505 3.750 5 Flow Rate 1.5 mL/min Run Time 5.800 min Elution orderSubstrate Set #14-(amide 14), ethyl-4-indole ester Column 25° CTemperature Injection Volume 10 μL Detection UV 230 and 260 nm;Detector: MWD

TABLE 12.7 Analytical Method Instrument Agilent 1200 Series HPLC withCTC-PAL Autosampler; AB Sciex 4000 Q-Trap MS Column Agilent Eclipse,C-18 4.6 × 50 mm, 1.6 μm Gradient I (A: 0.1% formic acid in water; B:0.1% formic acid in MeCN) Time(min) % A Mobile Phase 0.000 90 1.000 903.000  5 4.000  5 Flow Rate 0.6 mL/min Run Time 5.000 min MRM TargetMass MRM: 204.3 → 105.4; (amide 5) MRM: 235.4 → 86.4; (amide 15) ColumnRT Temperature Injection Volume 10 μL (Samples were quenched by 2-folddilution in 1:1 MeCN:water) Detection LC/MS/MS analysis ParametersSource dependent parameters: CUR: 40; IS: 5500; TEM: 550° C.; GS1: 40;G52: 40; DP: 120; EP: 10; CE: 27; CXP: 14.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. A polynucleotide sequence encoding at least one engineeredcarboxyesterase comprising a polypeptide sequence having at least 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to SEQ ID NO: 2 or a functional fragmentthereof, wherein said engineered carboxyesterase comprises at least onesubstitution or substitution set in said polypeptide sequence, whereinthe amino acid positions of said polypeptide sequence are numbered withreference to SEQ ID NO:
 2. 2. The polynucleotide sequence of claim 1,wherein said encoded at least one engineered carboxyesterase comprisesat least one substitution or substitution set at positions selectedfrom: 39, 39/323, 62, 62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263,77, 77/184, 103, 103/147, 104, 104/429, 105, 107, 107/185, 108, 109,109/117, 110, 111, 113, 114, 115, 117, 118, 118/269, 118/349, 119, 126,147, 153, 153/215, 164, 164/271, 174, 174/282, 183, 184, 184/249, 185,186, 187, 188, 190, 209, 210, 211, 212, 213, 213/271, 213/345, 214, 215,215/271, 216, 217, 217/231, 224, 224/268/372, 231, 249, 249/284, 263,268, 269, 270, 270/470, 271, 271/416, 276, 277, 278, 279, 279/280/282,280, 281, 281/374, 282, 283, 283/429, 284, 284/438, 285, 286, 311, 317,320, 320/323, 320/323/372, 320/372/376, 320/376/377, 321, 323, 324, 345,349, 372, 372/376, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429,438, and 470, wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO:
 2. 3. Apolynucleotide sequence encoding at least one engineered carboxyesterasecomprising a polypeptide sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 8 or a functional fragment thereof,wherein said engineered carboxyesterase comprises at least onesubstitution or substitution set in said polypeptide sequence, whereinthe amino acid positions of said polypeptide sequence are numbered withreference to SEQ ID NO:
 8. 4. The polynucleotide sequence of claim 3,wherein said encoded engineered carboxyesterase comprises at least onesubstitution or substitution set in said polypeptide sequence atpositions selected from: 63, 63/65/108, 63/65/108/189, 63/65/108/377,63/65/282/285/320/323, 63/65/320/323, 63/108, 63/108/282/285/377,63/108/285/377, 63/108/320/323, 63/189, 63/212/215,63/212/215/268/269/343, 63/215, 63/215/269, 63/215/270/271, 63/215/343,63/268/269/270/429, 63/377, 65/69/70/281/372, 65/69/70/372, 65/69/372,65/70/372, 65/320, 65/320/323, 68, 68/69/189/214, 68/69/189/214/215,68/69/189/214/215/271, 68/69/189/214/215/271/281/282/343/381,68/69/189/214/271/280, 68/69/189/214/372, 68/69/189/214/377/381,68/69/189/271, 68/69/189/271/280/372/381, 68/69/189/280/281/282/372/377,68/69/189/281/282/372/377/381, 68/69/189/343/381, 68/69/189/372,68/69/189/381, 68/69/214/215/271, 68/69/214/343, 68/69/215, 68/69/271,68/69/282/287, 68/69/343/372, 68/108/377, 68/184, 68/184/189,68/189/271/372, 68/189/343, 68/214/215/271/281/282/372,68/215/271/343/372/381, 68/215/377, 68/271/372, 68/377, 69, 69/70,69/70/331/372, 69/70/372, 69/70/459, 69/108, 69/108/270/372/377,69/108/281/285, 69/110/215/281, 69/189, 69/189/214/271/281/282/343,69/189/214/343/372, 69/189/215/343, 69/189/271, 69/189/271/281/282,69/189/271/343, 69/189/271/343/381, 69/189/280/282/343/372/381,69/189/281/373, 69/189/282, 69/189/343/381, 69/189/372, 69/189/372/377,69/189/377, 69/212/213/215/280/281, 69/214/215/271/372/377/381,69/214/271/282, 69/214/271/343, 69/215, 69/215/269/270/377,69/215/271/280/281/282, 69/215/271/282, 69/215/271/372, 69/215/285/317,69/215/323, 69/215/343, 69/215/343/372/381, 69/271, 69/271/372, 69/282,69/282/343/372, 69/285/373, 69/372, 70, 70/212, 108, 108/189,108/189/282/285/320, 108/189/320, 108/189/377, 108/215, 108/215/377,108/269/270, 108/270, 108/282/285/377, 108/285, 108/320/323, 108/377,126, 126/184/213/280/281/285/320, 126/184/213/372, 126/189/285/372,126/215, 126/372, 181/215, 189, 189/214, 189/214/215/271/282, 189/215,189/215/249/277, 189/215/271/281/282/377, 189/215/343/372, 189/270/285,189/270/372, 189/280/282, 189/320/377, 189/343, 189/343/377,189/372/377, 189/377, 189/381, 213/215/320, 214/215/271,214/215/271/377, 214/271, 214/280/282/343/377/381, 215,215/249/280/281/285/372, 215/271/372, 215/280/281/285/372,215/281/285/372, 215/281/285/373, 215/281/373, 215/285, 215/285/317,215/285/346, 215/285/445, 215/320, 215/320/372, 215/323, 215/372,215/372/377, 215/373, 215/377, 215/381, 249/377, 269/270/281/372/377,270/377, 271, 271/343, 271/343/372, 271/343/372/381, 280/285/372,281/372, 282/285/320/323, 285/323, 320, 343/372, 372, 372/377, 372/381,373, and 377, wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO:
 8. 5. Apolynucleotide sequence encoding at least one engineered carboxyesteraseor a functional fragment thereof, said polynucleotide sequencecomprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%, sequence identity to SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, and/or139.
 6. The polynucleotide sequence of claim 1, wherein saidpolynucleotide sequence is operably linked to a control sequence.
 7. Thepolynucleotide sequence of claim 1, wherein said polynucleotide sequenceis codon optimized.
 8. An expression vector comprising at least onepolynucleotide sequence of claim
 1. 9. A host cell comprising at leastone expression vector of claim
 8. 10. A host cell comprising at leastone polynucleotide sequence of claim
 1. 11. The host cell of claim 9,wherein said host cell is E. coli.
 12. A method of producing anengineered carboxyesterase in a host cell, comprising culturing a hostcell comprising at least one polynucleotide of claim 1, under suitableconditions, such that at least one engineered carboxyesterase isproduced.
 13. The method of claim 12, further comprising recovering atleast one engineered carboxyesterase from the culture and/or host cell.14. The method of claim 13, further comprising the step of purifyingsaid at least one engineered carboxyesterase.